Monoclonal antibody to CEA, conjugates comprising said antibody, and their therapeutic use in an adept system

ABSTRACT

The invention relates to an anti-CEA monoclonal antibody (named “806.077 antibody”) of murine origin and useful for the diagnosis and therapy of cancer. The antibody complementarity determining regions (CDRs) have the following sequences: heavy chain; CDR1 DNYMH, CDR2 WIDPENGDTE YAPKFRG, CDR3 LIYAGYLAMD Y; and light chain CDR1 SASSSVTYMH, CDR2 STSNLAS, CDR3 QQRSTYPLT. Humanised antibodies are described. The antibody is preferably in the form of a conjugate with either an enzyme suitable for use in an ADEPT system, especially a carboxypeptidase, or with a co-stimulatory molecule such as the extracellular domain of human B7.1.

[0001] The present invention relates to a novel anti-CEA monoclonalantibody (named “806.077 antibody” or “806.077 Ab” herein) useful forthe diagnosis and therapy of cancer.

[0002] It is established that the transformation of normal tissue cellsto tumour cells is associated with a change in structure on the cellsurface. Altered cell surface structures can serve as antigens and thetumour modified structures represent a type of so-calledtumour-associated antigen (see for example Altered Glycosylation inTumour Cells, Eds. Reading, Hakamori and Marcus 1988, Arthur R. Lisspubl.). Such antigens may be exploited for example by the generation ofmonospecific antibodies using hybridoma technology as is presently wellestablished after being first described by Kohler and Milstein (Nature,256, 495-497, 1975).

[0003] One tumour-associated antigen is CEA (Carcinomembryonic Antigen)as first described by Gold and Freedman, J Exp Med, 121, 439, 1965. Thisantigen is present on the tumour cell surface and can also bedemonstrated in blood serum.

[0004] The concept of using antibodies to target tumour associatedantigens in the treatment of cancer has been appreciated for some time(Herlyn et. al. (1980) Cancer Research 40, 717). Antibodies may be usedto target various chemical and biological agents to the tumour and suchconjugates have been particularly successful in forming the basis formany methods of both in vitro and in vivo diagnosis. The use ofimmunoconjugates in the therapy of cancer is also promising (Lord etal.(1985) Trends in Biotechnology 3, 175; Vitetta et al (1987) Science238, 1098). This approach is technically more demanding than diagnosticapplications and requires that tumour associated antigens which aretargetted in such immunotherapeutic approaches, are highly tumourspecific and not expressed at significant levels in vital human tissues.Whilst not wishing to be bound by theoretical considerations, as well asthe property of having specific tumour associated tissue distribution,for some applications it is desirable that the antibody remain at thecell surface after antigen binding rather than being quicklyinternalised. For example in ADEPT (antibody directed enzyme prodrugtherapy, see U.S. Pat. Nos. 4,975,278 and 5,405,990) it is believed tobe preferred that the antibody remain at the cell surface to facilitateprodrug activation by antibody-enzyme conjugate.

[0005] Antibody conjugates also have application in tumourimmunotherapy. The following few paragraphs set out the scientificbackground for this application. In order to respond to an immunestimulus, T-cells require two signals. One such signal is provided byrecognition of MHC displayed peptides by the T-cell receptor (TCR). Ithas been demonstrated however that TCR stimulation alone results inT-cell unresponsiveness or anergy and a second or co-stimulatory signalis required to stimulate specific T-cell activation and proliferation(reviewed by Schwartz R. H. J. Exp. Med., 1996, 184, 1-8). Uponreceiving both signals, the resulting cytotoxic T-cells mediate theimmune response by killing the target cells. A number of potentialco-stimulatory molecules have been identified (eg B7, ICAMs, LFA-1 and3, CD40, CD70 and CD24, reviewed by Galea-Lauri J. et al Cancer GeneTherapy, 1996, 3, 202-213). The major co-stimulatory function appears tobe provided by the related molecules B7.1 (also called CD80) and B7.2(also called CD86) which can interact with two receptors, CD28 andCTLA-4 (Hellstrom K. E. et al Immunol. Rev., 1995, 145, 123-145 andLenschow D. J. et al Ann. Rev. Immunol., 1996, 14, 233-258). B7.1 andB7.2 are expressed on antigen presenting cells (APC) such as dendriticcells whereas CD28 and CTLA-4 are present on T-cells. B7.2 appears to beconstitutively expressed on the surface of APCs but after contact with aT-cell, expression of B7.1 is up-regulated. Analogously, CD28 isexpressed on T-cells but after activation is down-regulated and replacedby CTLA4 expression. The stimulation of CD28 and CTLA-4 by B7.1 and B7.2represents a complex pattern of signalling which controls not only theactivation of the T-cell, but the subsequent control of proliferation tomodulate the immune-response (Greene J. et al J. Biol. Chem., 1996, 271,26762-26771). This phenomenon may explain the sometimes conflicting datareported by workers studying these co-stimulatory molecules.

[0006] In cancer, tumour infiltrating lymphocytes have been identifiedbut the lack of immune-response to the tumour may be due to T-cellanergy. Tumour cells can display specific or selective antigens on theirsurface but lack B7.1/B7.2 allowing them to escape immune surveillance.Indeed, in vivo experiments have demonstrated that B7.1/B7.2 transfectedtumour cells are less tumourigenic than untransfected cells from thesame line and that the transfected cells are capable of inducingprotective immunity against rechallenge with parental cells (Townsend S.E. and Allison J. P., 1993, Science, 259, 368-370). This demonstratesthat once stimulated, the immune response can become B7.1/B7.2independent. Hellstrom has proposed that expression of B7.1/B7.2 intumour cells by gene therapy has the potential to stimulate a hostreponse which can reduce or eliminate the disease. Gajewski (J.Immunol., 1996, 156, 465-472) and Matulonis et al (J. Immunol., 1996,156, 1126-1131) have reported that B7.1 is superior to B7.2 in theactivation of T-cells. The use of B7.1 in solution (as a fusion withantibody constant domains) is reported to provide only modestco-stimulation to T-cells receiving TCR stimulation via an independentsource (Linsley P. S. et al J. Exp. Med., 1991, 173, 721-730).

[0007] There is a need for further and improved anti-CEA antibodiesuseful in cancer diagnosis and therapy.

[0008] The present invention is based on the discovery of a novelanti-CEA antibody termed 806.077 antibody herein.

[0009] According to one aspect of the present invention there isprovided an anti-CEA antibody comprising complementarity determiningregions (CDRs) in which the CDRs have the following sequences: a) heavychain CDR1 DNYMH (SEQ ID NO: 29) CDR2 WIDPENGDTE YAPKFRG (SEQ ID NO: 31)CDR3 LIYAGYLAMD Y(SEQ ID NO: 32); b) light chain CDRl SASSSVTYMH (SEQ IDNO: 26) CDR2 STSNLAS (SEQ ID NO: 27) CDR3 QQRSTYPLT (SEQ ID NO: 28).

[0010] The CDRs or complementarity determining regions are thosesequences within the hypervariable loops of antibody variable domainswhich are believed to be critical in determining the specificity of theantigen-antibody interaction (Kabat, E. A., Lu, T. T., Reid-Miller, M.,Perry, H. M. & Gottesman, K. S. (1987). Sequences of Proteins ofImmunological Interest. 4th edition. Washington D.C.: United StatesDept. of Health and Human Services; the reader is also referred to thisreference for details of Kabat antibody residue numbering). CDRs asdefined herein however include framework residues where these contributeto binding. For the 806.077 antibody the CDRs were determined byhomology with the hypervariable sequences of other murine antibodies. Inthis specification the terms “VK” and “VH” mean variable regions of thelight and heavy antibody chains respectively. Anatomy of the antibodymolecule has been reviewed by Padlan (1994) in Molecular Immunology 31,169-217. The Light Chain CDRs are: VK CDR1 Kabat residues 24-34inclusive, SASSSVTYMH (SEQ ID NO: 26); VK CDR2 Kabat residues 50-56inclusive, STSNLAS (SEQ ID NO: 27); VK CDR3 Kabat residues 89-97inclusive, QQRSTYPLT (SEQ ID NO: 28); The Heavy Chain CDRs are: VH CDR1Kabat residues 31-35B inclusive, DNYMH (SEQ ID NO: 29); preferred VHCDR1 Kabat residues are no. 27-35B inclusive, FNIKDNYMH (SEQ ID NO: 30);VH CDR2 Kabat residues 50-65 inclusive, WIDPENGDTE YAPKFRG (SEQ ID NO:31) VH CDR3 Kabat residues 95-102 inclusive, LIYAGYLAMD Y (SEQ ID NO:32); and preferred VH CDR3 Kabat residues are no. 93-102 inclusive,HYLIYAGYLA MDY (SEQ ID NO: 33).

[0011] Preferably binding affinity for CEA antigen is at least 10E-5M,more preferably binding affinity for CEA is at least 10E-6M, morepreferably binding affinity for CEA is at least 10E-7M, more preferablybinding affinity for CEA is at least 10E-8M, more preferably bindingaffinity for CEA is at least 10E-9M, more preferably binding affinityfor CEA is at least 10E-10M and especially binding affinity for CEA isat least 10E-11M.

[0012] The term antibody as used herein generally means animmunoglobulin molecule (or fragment thereof or modified antibodyconstruct such as scFv which retains specific CEA antigen binding). TheCDRs are principally responsible for antigen binding, the non-CDRprotein sequence is normally derived from an immunoglobulin but may bederived from immunoglobulin domain of a immunoglobulin super familymember.

[0013] According to another aspect of the present invention there isprovided a CEA antibody comprising the following, optionally humanised,structure: a heavy chain variable region sequence (SEQ ID NO: 11)EVQLQQSGAE LVRSGASVKL SCTASGFNIK DNYMHWVKQR 40 PEQGLEWIAW IDPENGDTEYAPKFRGKATL TADSSSNTAY 80 LHLSSLTSED TAVYYCHVLI YAGYLAMDYW GQGTSVAVSS 120and; a light chain variable region sequence (SEQ ID NO: 9): DIELTQSPAIMSASPGEKVT ITCSASSSVT YMHWFQQKPG 40 TSPKLWIYST SNLASGVPAR FSGSGSGTSYSLTISRMEAE 80 DAATYYCQQR STYPLTFGAG TKLELKRA 108;

[0014] or any one of the following constructs thereof:

[0015] F(ab′)₂; F(ab′), Fab, Fv, single chain Fv & V-min.

[0016] F(ab′)₂ fragment constructs are preferred. Any suitable antibodyfragment which retains 806.077 antibody binding characteristics iscontemplated. For example a recently described antibody fragment is“L-F(ab)₂” as described by Zapata (1995) in Protein Engineering, 8,1057-1062. Disulphide bonded Fvs are also contemplated. Optionally theantibody forms part of a conjugate as described below.

[0017] A preferred humanised antibody comprises at least one of thefollowing sequences:

[0018] a heavy chain variable region sequence which is VH1 (SEQ ID NO:55);

[0019] a light chain variable region sequence which is VK4 (SEQ ID NO:71);

[0020] a human CH1 heavy chain IgG3 constant region;

[0021] a human kappa light chain CL region; and

[0022] a human IgG3 hinge region;

[0023] optionally in the form of a F(ab′)₂ fragment.

[0024] According to another aspect of the present invention there isprovided a polynucleotide sequence capable of encoding for the heavy orlight chain variable region of a CEA antibody of the invention.Preferably the heavy or light chain variable region is fused (optionallyvia some linking sequence) to a gene encoding a protein effector moiety(as part of a conjugate, see text below), preferably fusion is throughthe antibody heavy chain. Generally fusion can be either at the N or Cterminus of the antibody chain. For B7 conjugates fusion at theN-terminus of the antibody chain is preferred.

[0025] CPB has an N-terminal pro domain which is believed to assistcorrect folding of protein before the pro domain is removed to releaseactive enzyme. If proCPB is fused at its C terminus to the N terminus ofan antibody chain this allows removal of pro domain (e.g. by trypsintreatment) from the N terminus of the fission construct. Alternativelyif proCPB was attached to the C terminus of an antibody chain then theproblem arises of having to remove the pro domain from the “middle” ofthe construct without destroying the fusion protein. The solution is toco-express the pro domain separately (in trans). This has the advantage,once the cell lines have been constructed, of not requiring trypsinactivation of expressed fusion protein to remove CPB pro domain.Constructs with proCPB fused at its C terminus to the N terminus of anantibody chain have the advantage of not requiring construction ofco-expression cell lines which require high level expression of the prodomain along with high level expression of other proteins..

[0026] In this specification conservative amino acid analogues ofspecific amino acid sequences are contemplated which retain the bindingproperties of the CEA antibody of the invention but differ in sequenceby one or more conservative amino acid substitutions, deletions oradditions. However the specifically listed amino acid sequences arepreferred. Typical conservative amino acid substitutions are tabulatedbelow.

Conservative Substitutions

[0027] Original Exemplary Substitutions Preferred Substitutions Ala (A)Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His; Lys; ArgGln Asp (D) Glu Glu Cys (C) Ser Ser Gln (Q) Asn Asn Glu (E) Asp Asp Gly(G) Pro Pro His (H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val; Met; Ala;Phe; Leu Norleucine Leu (L) Norleucine; Ile; Val; Met; Ile Ala; Phe Lys(K) Arg; Gln; Asn Arg Met (M) Leu; Phe; Ile Leu Phe (F) Leu; Val; Ile;Ala Leu Pro (P) Gly Gly Ser (S) Thr Thr Thr (T) Ser Ser Tyr (Y) Trp;Phe; Thr; Ser Phe Val (V) Ile; Leu; Met; Phe: Ala; Leu Norleucine

[0028] In this specification nucleic acid variations (deletions,substitutions and additions) of specific nucleic acid sequences arecontemplated which retain which the ability to hybridise under stringentconditions to the specific sequence in question. A hybridisation test isset out in Example 9 hereinafter. However specifically listed nucleicacid sequences are preferred. It is contemplated that peptide nucleicacid may be an acceptable equivalent of polynucleotide sequences, atleast for purposes that do not require translation into protein (Wittung(1994) Nature 368, 561).

[0029] According to another aspect of the present invention there isprovided an antibody or antibody fragment as herein describedcharacterised in that it is humanised.

[0030] A humanised antibody, related fragment or antibody bindingstructure is a polypeptide composed largely of a structural framework ofhuman derived immunoglobulin sequences supporting non human derivedamino acid sequences in and around the antigen binding site(complementarity determining regions or CDRs; this technique is known asCDR grafting which often involves some framework changes too, see theExamples below). Appropriate methodology has been described for examplein detail in WO 91/09967, EP 0328404 and Queen et al. Proc Natl Acad Sci86, 10029, Mountain and Adair (1989) Biotechnology and GeneticEngineering Reviews 10, 1 (1992) although alternative methods ofhumanisation are also contemplated such as antibody veneering of surfaceresidues (EP 519596, Merck/NIH, Padlan et al). Preferred humanised806.077 antibodies are any one of Examples 11-47 or 107-122. A preferredhumanised heavy chain variable region is VH1 (see Examples). A preferredlight chain variable region is VK4 optionally incorporating any of theadditional changes described in Examples 107-109. A preferred humanheavy chain constant region is IgG3.

[0031] Chimaeric humanised antibodies represent another aspect of theinvention. Preparation of chimaeric humanised antibody fragments ofantibody 806.077 antibody is described in Example 8 herein. Chimaericantibodies are generally constructed by combining the variable regionfrom one species with a constant region from another antibody from adifferent species.

[0032] The term “humanised” in relation to antibodies as used hereinincludes any method of humanisation such as for example CDR grafting orchimaeric antibody preparation or any hybrid thereof such as for examplea CDR grafted heavy chain in combination with a chimaerised light chain(see Example 110 for a suitable embodiment).

[0033] In particular, a rodent antibody on repeated in vivoadministration in man either alone or as a conjugate will bring about animmune response in the recipient against the rodent antibody; theso-called HAMA response (Human Anti Mouse Antibody). The HAMA responsemay limit the effectiveness of the pharmaceutical if repeated dosing isrequired. The immunogenicity of the antibody may be reduced by chemicalmodification of the antibody with a hydrophilic polymer such aspolyethylene glycol or by using the methods of genetic engineering tomake the antibody binding structure more human like. For example, thegene sequences for the variable domains of the rodent antibody whichbind CEA can be substituted for the variable domains of a human myelomaprotein, thus producing a recombinant chimaeric antibody. Theseprocedures are detailed in EP 194276, EP 0120694, EP 0125023, EP0171496, EP 0173494 and WO 86/01533. Alternatively the gene sequences ofthe CDRs of the CEA binding rodent antibody may be isolated orsynthesized and substituted for the corresponding sequence regions of ahomologous human antibody gene, producing a human antibody with thespecificity of the original rodent antibody. These procedures aredescribed in EP 023940, WO 90/07861 and WO91/09967. Alternatively alarge number of the surface residues of the variable domain of therodent antibody may be changed to those residues normally found on ahomologous human antibody, producing a rodent antibody which has asurface ‘veneer’ of residues and which will therefore be recognized asself by the human body. This approach has been demonstrated by Padlanet. al. (1991) Mol. Immunol. 28, 489.

[0034] According to another aspect of the present invention there isprovided a host cell transformed with a polynucleotide sequence or atransgenic non-human animal or transgenic plant developed from the hostcell in which the polynucloetide sequence encodes at least the variableregion of the heavy chain or light chain of a CEA antibody of theinvention, optionally in the form of a conjugate as described herein.

[0035] According to another aspect of the present invention there isprovided hybridoma 806.077 deposited as ECACC deposit no. 96022936 andvariant cell lines thereof.

[0036] Hybridoma 806.077 antibody was deposited at the EuropeanCollection of Animal Cell Cultures (ECACC), PHLS Centre for AppliedMicrobiology & Research, Porton Down, Salisbury, Wiltshire SP4 0JG,United Kingdom on Feb. 29, 1996 under accession no. 96022936 inaccordance with the Budapest Treaty.

[0037] According to another aspect of the present invention there isprovided plasmid pNG3-Vkss-HuCk deposited as NCIMB deposit no. 40798.

[0038] Plasmid pNG3-Vkss-HuCk was deposited at The National Collectionsof Industrial and Marine Bacteria (NCIMB), 23 St Machar Drive, AberdeenAB2 1RY, Scotland, United Kingdom on Apr. 11, 1996 under depositreference number NCIMB 40798 in accordance with the Budapest Treaty.

[0039] According to another aspect of the present invention there isprovided plasmid pNG4-VHss-HuIgG2CH1′ deposited as NCIMB deposit no.40797.

[0040] Plasmid pNG4-VHss-HuIgG2CH1′ was deposited at The NationalCollections of Industrial and Marine Bacteria (NCIMB), 23 St MacharDrive, Aberdeen AB2 1RY, Scotland, United Kingdom on Apr. 11, 1996 underdeposit reference number NCIMB 40797 in accordance with the BudapestTreaty.

[0041] According to another aspect of the present invention there isprovided plasmid pNG3-Vkss-HuCk-NEO deposited as NCIMB deposit no.40799.

[0042] Plasmid pNG3-Vkss-HuCk-NEO was deposited at The NationalCollections of Industrial and Marine Bacteria (NCIMB), 23 St MacharDrive, Aberdeen AB2 1RY, Scotland, United Kingdom on Apr. 11, 1996 underdeposit reference number NCIMB 40799 in accordance with the BudapestTreaty.

[0043] According to another aspect of the present invention there isprovided a method of making at least a variable region of a heavy orlight chain of an anti-CEA antibody as herein defined comprising:

[0044] a) transforming a host cell with a polynucleotide sequence whichencodes at least the variable region of the heavy or light chain of theanti-CEA antibody and optionally developing the transformed host cellinto a transgenic non-human mammal or transgenic plant;

[0045] b) subjecting the host cell, transgenic non-human mammal ortransgenic plant to conditions conducive to expression, and optionallysecretion, of at least the variable region and optionally;

[0046] c) at least partially purifying the variable region.

[0047] According to another aspect of the present invention there isprovided a method of making an antibody or a conjugate as defined hereinwhich comprises:

[0048] a) subjecting a host cell, a transgenic non-human mammal or atransgenic plant as defined herein, or the 806.077 hybridoma, toconditions conducive to expression, and optionally secretion, of theantibody or conjugate; and optionally

[0049] b) at least partially purifying the antibody or conjugate.

[0050] Preferably both heavy and light chain variable regions areexpressed in the same cell and assembled thereby to form an anti-CEAantibody. Preferably the heavy or light chain variable region is fused(optionally via some linking sequence) to a gene encoding a proteineffector moiety (as part of a conjugate, see text below), preferablyfusion is through the antibody heavy chain. Generally fusion can beeither at the N or C terminus of the antibody chain. For B7 conjugatesfusion at the N-terminus of the antibody chain is preferred. CPB has anN-terminal pro domain which is believed to assist correct folding ofprotein before the pro domain is removed to release active enzyme. IfproCPB is fused at its C terminus to the N terminus of an antibody chainthis allows removal of pro domain (e.g. by trypsin treatment) from the Nterminus of the fusion construct. Alternatively if proCPB was attachedto the C terminus of an antibody chain then the problem arises of havingto remove the pro domain from the “middle” of the construct withoutdestroying the fusion protein. The solution is to co-express the prodomain separately (in trans). This has the advantage, once the celllines have been constructed, of not requiring trypsin activation ofexpressed fusion protein to remove CPB pro domain. Constructs withproCPB fused at its C terminus to the N terminus of an antibody chainhave the advantage of not requiring construction of co-expression celllines which require high level expression of the pro domain along withhigh level expression of other proteins.

[0051] According to another aspect of the present invention there isprovided a method of making monoclonal antibody 806.077 comprising:

[0052] a) culturing hybridoma 806.077 antibody deposited as ECACCdeposit no. 96022936 in medium under conditions conducive to expressionof antibody therefrom and;

[0053] b) obtaining antibody 806.077 antibody from the culture mediumand optionally;

[0054] c) preparing a F(ab′)₂ fragment of antibody 806.077 antibody byenzymic digestion.

[0055] According to another aspect of the present invention there isprovided a conjugate which comprises an effector moiety and an anti-CEA806.077 antibody of the invention as herein described. An effectormoiety is an entity having the effect of bestowing another activity(e.g. an enzyme, toxin or radioactive ligand) to the 806.077 antibody informing the conjugate.

[0056] In one embodiment, preferably the effector moiety is an enzymesuitable for use in an ADEPT system. In International Patent ApplicationWO 96/20011, published Jul. 4, 1996, we proposed a “reversed polarity”ADEPT system based on mutant human enzymes having the advantage of lowimmunogenicity compared with for example bacterial enzymes. A particularhost enzyme was human pancreatic CPB (see for example, Example 15[D253K]human CPB & 16 [D253R]human CPB therein) and prodrugs therefor(see Examples 18 & 19 therein). The host enzyme is mutated to give achange in mode of interaction between enzyme and prodrug in terms ofrecognition of substrate compared with the native host enzyme. In oursubsequent International Patent Application No PCT/GB96/01975 (publishedMar. 6, 1997 as WO 97/07796) further work on mutant CPB enzyme/prodrugcombinations for ADEPT are described. Preferred enzymes suitable forADEPT are any one of CPG2 or a reversed polarity CPB enzyme, for exampleany one of [D253K]HCPB, [G251T,D253K]HCPB or [A248S,G251T,D253K]HCPB.

[0057] 806.077 Antibody conjugates also have application in tumourimmunotherapy. Accordingly in another preferred embodiment the conjugateeffector moiety is a co-stimulatory molecule, preferably theco-stimulatory molecule is B7, more preferably human B7.1 or B7.2 andespecially human B7.1. Preferably the conjugate is in the form of afusion protein, preferably in which the fusion protein is formed throughlinking a C-terminus of the co-stimulatory molecule to an N-terminus806.077 antibody chain, preferably via the antibody chain heavy chain,preferably in which the 806.077 antibody lacks an Fc antibody region,more preferably a F(ab′)₂ antibody fragment, more preferably theantibody is humanised or human. An especially preferred conjugate isdescribed in Example 104 below.

[0058] The use of antibody to target a co-stimulatory molecule to tumourcells is predicted to bestow the function of antigen presenting to thetumour cells such that T-cells receive specific TCR stimulation from thetumour cell itself and a co-stimulatory signal from the antibodytargeted molecule. The use of human or humanised antibodies is preferredfor the treatment of human tumours because murine antibodies may evokean immune reaction when used in man which might result in a reduction ineffectivness on repeat therapy. The use of a fusion protein combining atumour antigen binding region linked to the extracellular portion of aco-stimulatory molecule is novel. Hayden et al (Tissue Antigens, 1996,48, 242-254) have reported the use of a bi-specific antibody moleculecombining an anti-tumour antigen binding domain with an anti-CD28binding domain. Whilst this molecule is capable of interacting with CD28on T-cells, the signal it may deliver has the disadvantage of beingqualitatively different from that provided by the natural CD28 ligands,for example the affinity of binding is greater than that between B7.1and CD28. The cross-species homology between B7.1, B7.2 and CD28, CTLA4indicates evolutionary conservation of binding region sequences.Consequently it is believed that, for example B7.1 from man can interactwith CD28 from mouse and may impart a similar co-stimulatory signal. Fortreatment of human disease a human or humanised protein is preferable.However, the use of a human or humanised protein in animal models couldproduce similar effects to that anticipated in man and such animalmodels should provide relevant data as to the efficacy of ahuman/humanised antibody fusion protein with human B7.1/B7.2 in thetreatment of human disease.

[0059] Conjugation of the effector moiety and antibody may be by anysuitable method such as for example chemical linkage viaheterobifunctional linkers or recombinant gene fusion techniques. Ingeneral fusion proteins are preferred conjugates, particularly forconjugates with HCPB or B7.

[0060] Preferred conjugates are those in which the effector moiety isselected from any one of the following:

[0061] a) an enzyme suitable for use in an ADEPT system;

[0062] b) CPG2;

[0063] c) [G251T,D253K]HCPB;

[0064] d) [A248S,G251T,D253K]HCPB;

[0065] e) a co-stimulatory molecule;

[0066] f) extracellular domain of B7;

[0067] g) extracellular domain of human B7.1; and

[0068] h) extracellular domain of human B7.2;

[0069] optionally in the form of a fusion protein.

[0070] It will be appreciated that the conjugate of the presentinvention does not necessarily consist of one effector molecule and oneantibody molecule. For example the conjugate may comprise more than oneeffector molecule per antibody molecule. In general, F(ab′)₂ antibodyconjugates which are fusions between the antibody and an enzyme or anextracellular domain of B7 will have 2 moles of enzyme or B7 per mole ofantibody.

[0071] Especially preferred conjugates are a fusion protein selectedfrom any one of the following conjugates, (sequences being listed in Nterminus to C terminus direction):

[0072] a) a humanised 806.077 F(ab′)₂-{[A248S,G251T,D253K]HCPB}₂ fusioncomprising:

[0073] an antibody Fd′ chain of structure VH1(SEQ ID NO: 55)/CH1constant region from IgG3/hinge region from IgG3;

[0074] the Fd′ chain being fused via its C terminus to the N terminus of[A248S,G251T,D253K]HCPB; and

[0075] an antibody light chain of formula VK4(SEQ ID NO: 71)/CL regionfrom kappa light chain;

[0076] b) {[A248S,G251T,D253K]HCPB}₂-humanised 806.077 F(ab′)₂ fusioncomprising:

[0077] [A248S,G251T,D253K]HCPB;

[0078] the HCPB being fused at its C terminus, via a (GGGS)₃ linker, tothe N terminus of an antibody Fd′ chain of structure VH1(SEQ ID NO:55)/CH1 constant region from IgG3/hinge region from IgG3; and

[0079] an antibody light chain of formula VK4(SEQ ID NO: 71)/CL regionfrom kappa light chain; and

[0080] c) a (human B7.1 extracellular domain)₂—humanised 806.077 F(ab′)₂fusion comprising:

[0081] human B7.1 extracellular domain;

[0082] the B7.1 being fused at its C terminus to the N terminus of anantibody Fd′ chain of structure VH1(SEQ ID NO: 55)/CH1 constant regionfrom IgG3/hinge region from IgG3: and

[0083] an antibody light chain of structure VK4(SEQ ID NO: 71)/CL regionfrom kappa light chain.

[0084] In this specification the antibody hinge region in relation toconjugates is defined according to the principles set out by Padlan(1994) in Molecular Immunology 31, 169-217: see Table 2 therein inparticular. In these especially preferred conjugates there are 2 molesof enzyme or B7.1 per mole of F(ab′)₂. The forward slash (“/”) is merelya separator to indicate discrete structural elements joined by peptidebonds that make up parts of the conjugate.

[0085] The VH1 and/or VK4 variable region humanised sequences have theadvantage of preserving good binding properties with minimal additionalchanges required to the human framework. The IgG3 hinge region has theadvantage of giving good F(ab′)₂ production levels and homogeneity ofproduct.

[0086] In another preferred embodiment relating to the especiallypreferred conjugates defined above, fusion is effected through theantibody light chain. In yet another preferred embodiment relating tothe especially preferred conjugates defined above, the CH1 constantregion from IgG3/hinge region from IgG3 structural element may bereplaced by the corresponding IgG2 element.

[0087] When the effector molecule is a toxin, this toxin moietygenerally comprises a component which possesses cytotoxic properties andhence is capable of killing cells following internalisation.

[0088] The toxin moiety and the anti-CEA antibody may be coupleddirectly to one another, or they may be coupled indirectly. The toxinmoiety and the anti-CEA antibody are, in general, coupled such that thegeometry of the conjugate permits the anti-CEA antibody to bind to itstarget cell. Advantageously, the toxin moiety and the anti-CEA antibodyare coupled such that the conjugate is extracellularly stable, andintracellularly unstable so that the toxin moiety and the anti-CEAantibody remain coupled outside the target cell, but followinginternalisation, the toxin moiety is released. Thus, advantageously theconjugate has an intracellularly cleavable/extracellularly stable site.

[0089] Examples of conjugates in which the toxin moiety is directlycoupled to the target cell binding moiety include those in which thetoxin moiety and the anti-CEA antibody are coupled by a disulphidebridge formed between a thiol group on the toxin moiety and a thiolgroup on the anti-CEA antibody. Details of the preparation andproperties of immunotoxins and other conjugates are given in Europeanpatent application EP 528 527 (publication no.) the contents of which isincorporated herein by reference thereto.

[0090] According to another aspect of the present invention there isprovided a polynucleotide sequence capable of encoding a polypeptide ofan antibody or a conjugate as defined in any preceding claim. The term“capable of encoding” is intended to encompass polynucleotide sequencestaking into account degeneracy in the genetic code in that some aminoacids are encoded by more than one codon.

[0091] According to another aspect of the present invention there isprovided a vector comprising a polynucleotide as defined above.

[0092] According to another aspect of the present invention there isprovided a host cell transformed with a polynucleotide sequence asdefined above or a transgenic non-human animal or transgenic plantdeveloped from the host cell.

[0093] According to another aspect of the present invention there isprovided a pharmaceutical composition comprising a conjugate of theinvention described herein in association with apharmaceutically-acceptable diluent or carrier, optionally in a formsuitable for intravenous administration.

[0094] According to another aspect of the present invention there isprovided a conjugate as described herein for use as a medicament.

[0095] According to another aspect of the present invention there isprovided use of a conjugate as described herein for preparation of amedicament for treatment of neoplastic disease.

[0096] It will be appreciated that the dose and dosage regimen willdepend upon the particular effector moiety employed, the population ofthe target cell and the patient's history. The dose of the conjugateadministered will typically be in the range 0.1 to 1 mg/kg of patientweight.

[0097] The conjugates of the present invention will normally beadministered in the form of a pharmaceutical composition. Thus accordingto the present invention there is also provided a pharmaceuticalcomposition which comprises a conjugate (as defined herein) inassociation with a pharmaceutically-acceptable diluent or carrier. Anexample of such a formulation is given herein in Example 10.

[0098] Pharmaceutical compositions of the present invention may beformulated in a variety of dosage forms. Generally, the conjugates ofthe present invention will be administered parenterally, preferablyintravenously. A particular parenteral pharmaceutical composition is onewhich is formulated in a unit dosage form which is suitable foradministration by injection. Thus, particularly suitable compositionscomprise a solution, emulsion or suspension of the immunotoxin inassociation with a pharmaceutically acceptable parenteral carrier ordiluent. Suitable carriers or diluents include aqueous vehicles, forexample water or saline, and non-aqueous vehicles, for example fixedoils or liposomes. The compositions may include agents which enhance thestability of the conjugate in the composition. For example, thecomposition may include a buffer. The concentration of the conjugatewill vary, but in general, the conjugate will be formulated atconcentrations of about 1 to 10 mg/ml.

[0099] According to another aspect of the present invention there isprovided an expression vector coding for an anti-CEA antibody of theinvention as herein defined.

[0100] According to another aspect of the present invention there isprovided an expression vector encoding at least the variable region of aheavy or light chain of an anti-CEA antibody as herein defined.

[0101] According to another aspect of the present invention there isprovided a host cell transformed with a vector as herein described whichis compatible with expression therein.

[0102] According to another aspect of the present invention there isprovided a host cell transformed with a polynucleotide sequence asherein defined.

[0103] Mammalian cells (CHO, COS, myeloma) have been used as host forthe co-expression of antibody H and L chain cDNAs and fragments thereofto produce antibody with the specified binding activity (Bebbington, C.,1991, Methods, vol 2, p136-145, and Adair, J., 1992, ImmunologicalReviews, vol 130). For expression of constructs leading to directexpression of active CPB, COS or CHO cell expression systems arepreferred. The cDNAs can be introduced on plasmids and allowed tointegrate into chromosomal DNA especially for CHO cells or allowed toreplicate to very high copy number especially in COS cells. The plasmidsgenerally require a selectable marker for maintenance in transfectedhosts, an efficient eukaryotic promoter to allow a high level oftranscription from the cDNAs, convenient restriction enzyme sites forcloning and polyadenylation and transcription termination signals formessage stabilty. Several such vectors have been described in theliterature (Bebbington, C. et al, 1992, Bio/Technology, vol 10,p169-175, and Wright, A., 1991, Methods, vol 2, p125-135) and there arecommercially available vectors, (such as pRc/CMV, Invitrogen Corp.)which are suitable.

[0104] The expression of a range of antibody fragments in E. coli iswell documented (reviewed by Pluckthun, A., Immunological Reviews, 1992,vol 130, p151-188 and Skerra, A., Current Opinion in Immunology, 1993,vol 5, p256-262). Intracellular expression of Fd and L chains has beendescribed (Cabilly, S., 1989, Gene. vol 85, p553-557) but this mayrequire in vitro refolding and re-association of the chains (Buchner, Jand Rudolph, R., 1991, Bio/Technology, vol 9, p157-162) to producebinding activity. A more efficient route to obtaining active antibodyfragments is through periplasmic secretion (Better, M. et al, 1988,Science, vol 240, p1041-1043). The H and L chain components of theantibody fragment are co-expressed from a single plasmid. Each antibodychain is provided with a bacterial leader peptide which directs it tothe E. coli periplasm where the leader is cleaved and the free chainsassociate to produce soluble and active antibody fragments. This processis believed to mimic the natural process in eukaryotic cells where theexpressed antibody chains pass into the lumen of the endoplasmicreticulum prior to association into whole antibodies. This process oftenresults in the presence of binding activity in the culture supernatant.

[0105] Some expression systems involve transforming a host cell with avector; such systems are well known such as for example in E. coli,yeast and mammalian hosts (see Methods in Enzymology 185, Academic Press1990). Other systems of expression are also contemplated such as forexample transgenic non-human mammals in which the gene of interest,preferably cut out from a vector and preferably in association with amammary promoter to direct expressed protein into the animal's milk, isintroduced into the pronucleus of a mammalian zygote (usually bymicroinjection into one of the two nuclei (usually the male nucleus) inthe pronucleus) and thereafter implanted into a foster mother. Aproportion of the animals produced by the foster mother will carry andexpress the introduced gene which has integrated into a chromosome.Usually the integrated gene is passed on to offspring by conventionalbreeding thus allowing ready expansion of stock. Preferably the proteinof interest is simply harvested from the milk of female transgenicanimals. The reader is directed to the following publications: Simons etal. (1988), Bio/Technology 6:179-183; Wright et al. (1991)Bio/Technology 9:830-834; U.S. Pat. No. 4,873,191 and; U.S. Pat. No.5,322,775. Manipulation of mouse embryos is described in Hogan et al,“Manipulating the Mouse Embryo; A Laboratory Manual”, Cold Spring HarborLaboratory 1986.

[0106] Transgenic plant technology is also contemplated such as forexample described in the following publications: Swain W. F. (1991)TIBTECH 9: 107-109; Ma J. K. C. et al (1994) Eur. J. Immunology 24:131-138; Hiatt A. et al (1992) FEBS Letters 307:71-75; Hein M. B. et al(1991) Biotechnology Progress 7: 455-461; Duering K. (1990) PlantMolecular Biology 15: 281-294.

[0107] If desired, host genes can be inactivated or modified usingstandard procedures as outlined briefly below and as described forexample in “Gene Targeting; A Practical Approach”, IRL Press 1993. Thetarget gene or portion of it is preferably cloned into a vector with aselection marker (such as Neo) inserted into the gene to disrupt itsfunction. The vector is linearised then transformed (usually byelectroporation) into embryonic stem (ES) cells (eg derived from a129/Ola strain of mouse) and thereafter homologous recombination eventstake place in a proportion of the stem cells. The stem cells containingthe gene disruption are expanded and injected into a blastocyst (such asfor example from a C57BL/6J mouse) and implanted into a foster motherfor development. Chimaeric offspring can be identified by coat colourmarkers. Chimeras are bred to ascertain the contribution of the ES cellsto the germ line by mating to mice with genetic markers which allow adistinction to be made between ES derived and host blastocyst derivedgametes. Half of the ES cell derived gametes will carry the genemodification. Offspring are screened (eg by Southern blotting) toidentify those with a gene disruption (about 50% of progeny). Theseselected offspring will be heterozygous and therefore can be bred withanother heterozygote and homozygous offspring selected thereafter (about25% of progeny). Transgenic animals with a gene knockout can be crossedwith transgenic animals produced by known techniques such asmicroinjection of DNA into pronuclei, sphaeroplast fusion (Jakobovits etal. (1993) Nature 362:255-258) or lipid mediated transfection (Lamb etal. (1993) Nature Genetics 5 22-29) of ES cells to yield transgenicanimals with an endogenous gene knockout and foreign gene replacement.

[0108] ES cells containing a targeted gene disruption can be furthermodified by transforming with the target gene sequence containing aspecific alteration, which is preferably cloned into a vector andlinearised prior to transformation. Following homologous recombinationthe altered gene is introduced into the genome. These embryonic stemcells can subsequently be used to create transgenics as described above.

[0109] The term “host cell” includes any procaryotic or eucaryotic cellsuitable for expression technology such as for example bacteria, yeasts,plant cells and non-human mammalian zygotes, oocytes, blastocysts,embryonic stem cells and any other suitable cells for transgenictechnology. If the context so permits the term “host cell” also includesa transgenic plant or non-human mammal developed from transformednon-human mammalian zygotes, oocytes, blastocysts, embryonic stem cells,plant cells and any other suitable cells for transgenic technology.

[0110] According to another aspect of the present invention there isprovided a method of treatment of a human or animal in need of suchtreatment which comprises administration to a human or animal of apharmaceutically effective amount of a conjugate as herein described.

[0111] According to another aspect of the present invention there isprovided a method of targeting an effector moiety to cells displayingantigen CEA in a mammal in need of such targeting which comprisesadministration of a pharmaceutically effective amount of an conjugate ofthe invention as herein defined.

[0112] According to another aspect of the present invention there isprovided the use of an antibody as hereinbefore described in adiagnostic method.

[0113] One diagnostic method is immunoassay. An immunoassay for in vitrotesting based upon the novel antibody according to the invention may bedesigned in accordance with conventional immunological techniques in theart, utilising the antibody according to the invention in a labelled orunlabelled form and determining the complex formation of the antibodywith CEA in the sample to be tested. In one case, the antibody may belabelled with a detectable label, such as radiolabel, a chemiluminescer,a fluorescer or an enzyme label. Alternatively the antibody is detectedvia a complex formed with a labelled substance or by non-labellingtechniques, such as biosensor methods eg based upon surface plasmonresonance. The sample may, for example, be in the form of a body fluid,such as serum, or a tissue preparation (histochemical assay).

[0114] For in vivo diagnostic purposes, the antibody according to theinvention is provided with a suitable externally detectable label, suchas eg. a radiolabel or a heavy metal atom, and administered to a subjectwhereupon the possible localised accumulation of antibody in the body isdetermined.

[0115] For the in vitro diagnosis of cancer the anti-CEA antibody can beconjugated to either enzymes such as horse radish peroxidase andbacterial luciferase which can generate a signal which can be measuredor to fluorescent markers or radioisotopes which can be detected andquantitated directly. In a standard immunoassay system such conjugatesprovide a means of measuring the presence or absence of CEA in bodytissues and consequently provides a rapid and convenient test for thediagnosis of tumour disease. See general descriptions of the methodologyinvolved in Enzyme Immunoassay, E.T. Maggio, CRC Press and U.S. Pat. No.3,690,8334, U.S. Pat. No. 3,791,932, U.S. Pat. No. 3,817,837, U.S. Pat.No. 3,850,578, U.S. Pat. No. 3,853,987, U.S. Pat. No. 3,867,517, U.S.Pat. No. 3,901,654, U.S. Pat. No. 3,935,074, U.S. Pat. No. 3,984,533,U.S. Pat. No. 3,996,345 and U.S. Pat. No. 4,098,876.

[0116] For the in vivo diagnosis of cancer, the anti-CEA antibody can beconjugated to isotopes of elements such as yttrium, technetium or indiumor heavy metal isotopes which can be detected by whole body imagingcameras (see Larson, S. M., 1987, Radiology, 165, 297-304.

[0117] For the therapy of cancer, preferred embodiments involve ananti-CEA antibody that can be conjugated to an effector moiety which cankill the cancer cells directly or especially via activation of asuitable prodrug in an ADEPT system. In ADEPT selective killing oftumour cells is achieved by conjugating the anti-CEA antibody to anenzyme which is capable of catalysing the conversion of a non-toxic doseof a prodrug into a potent toxic drug compound. Administration of theconjugate leads to localization of the enzyme activity at the tumoursite. Subsequent administration of the prodrug leads to local productionof the toxic drug and selective kill at the tumour site. This approachis described in WO 88/07378, U.S. Pat. No. 4,975,278, U.S. Pat. No.5,405,990 and WO89/10140. Antibody 806.077 may also be used conjugatedto a co-stimulatory molecule for tumour immunotherapy as describedabove.

[0118] Selective cell killing of tumour cells can also be achieved byconjugation of the anti-CEA antibody either directly or by chemicalderivatization with macrocycle chelators containing high energyradioisotopes such as ⁹⁰Y, ¹³¹I and ¹¹¹In. The anti-CEA antibody servesto localize the isotope to the tumour and the radiation emitted by theisotope destroys the DNA of the surrounding cells and kills the tumour.

[0119] Selective killing of tumour cells can also be achieved byconjugation of the anti-CEA antibody to cytotoxic and cytostatic drugssuch as methotrexate, chlorambucil, adriamycin, daunorubicin andvincristine. These drugs have been used in the clinic for many years andthe therapy they provide is often limited by non specific toxicity.Conjugation of these drugs to the CEA antibody enables these drugs tolocalize at the tumour site and thus increasing the dose of drug thatcan be delivered to the tumour without incurring unacceptable sideeffects from the action of such drugs on other tissues such as the bonemarrow or nervous system.

[0120] The effectiveness of the antibody is in many applicationsimproved by reducing the size of the antibody binding structure andthereby improving the tissue penetration and other pharmacodynamicproperties of the pharmaceutical composition. This can be achieved byremoving the Fc region of the antibody molecule either enzymically or bygenetic engineering methods to produce a recombinant Fab′ or F(ab′)₂fragment.

[0121] Genetic engineering methods can also be used to further reducethe size of the anti-CEA antibody. The Fv which contain the CDRs can beengineered and expressed in isolation and chemically cross linked forinstance by the use of disulphide bridges. Alternatively, both the lightand heavy chain domains making up the Fv structure may be produced as asingle polypeptide chain (SCFv) by fusing the Fv domains with a linkerpeptide sequence from the natural C-terminus of one domain to theN-terminus of the other domain (see PCT/US/87/02208 and U.S. Pat. No.4,704,692). Alternatively, a single Fv domain may be expressed inisolation forming a single domain antibody or dAb as described by Wardet al Nature (1989) 341, 544. Another type of anti-CEA antibodycontemplated is a V-min construct as disclosed in International PatentApplication WO 94/12625 (inventors Slater & Timms).

[0122] Abbreviations used herein include: ADEPT antibody directed enzymeprodrug therapy APC antigen presenting cell CDRs complementaritydetermining regions CEA Carcinoma Embryonic Antigen CL constant domainof antibody light chain CPB carboxypeptidase B CPG2 carboxypeptidase G2DAB substrate 3,3′-diaminobenzidine tetrahydrochloride DEPCdiethylpyrocarbonate DMEM Dulbecco's modified Eagle's medium ECACCEuropean Collection of Animal Cell Cultures EIA enzyme immunoassay ELISAenzyme linked immunosorbent assay FCS foetal calf serum Fd heavy chainof Fab, Fab′ or F(ab′)₂ optionally containing a hinge HAMA Human AntiMouse Antibody HCPB human carboxypeptidase B, preferably pancreatichinge (of an IgG) a short proline rich peptide which contains thecysteines that bridge the 2 heavy chains HRPO horse radish peroxidaseNCA non-specific cross reacting antigen NCIMB National Collections ofIndustrial and Marine Bacteria PBS phosphate buffered saline PCRpolymerase chain reaction preproCPB proCPB with an N-terminal leadersequence proCPB CPB with its N-terminal pro domain SDS-PAGE sodiumdodecyl sulphate - polyacrylamide gel electrophoresis TBS Tris-bufferedSaline VH variable region of the heavy antibody chain VK variable regionof the light antibody chain

[0123] The invention is illustrated by the following non-limitingExamples (supported by Reference Examples which follow the Examples) inwhich:

[0124]FIG. 1 shows anti-tumour activity of 806.077 antibody-CPG2conjugate in an ADEPT model;

[0125]FIG. 2 shows a plasmid map of pCF009;

[0126]FIG. 3 shows BIAcore data showing antibody-B7.1 fusion proteinbinding to immobilised CTLA4-Ig in which the solid line represents testbinding and the dotted line is a blank control; and unless otherwisestated;

[0127] DNA is recovered and purified by use of GENECLEAN™ II kit(Stratech Scientific Ltd. or Bio 101 Inc.). The kit contains: 1) 6Msodium iodide; 2) a concentrated solution of sodium chloride, Tris andEDTA for making a sodium chloride/ethanol/water wash; 3) Glassmilk—a 1.5ml vial containing 1.25 ml of a suspension of a specially formulatedsilica matrix in water. This is a technique for DNA purification basedon the method of Vogelstein and Gillespie published in Proceedings ofthe National Academy of Sciences USA (1979) Vol 76, p 615. Briefly, thekit procedure is as follows. To 1 volume of gel slice is added 3 volumesof sodium iodide solution from the kit. The agarose is melted by heatingthe mix at 55° C. for 10 min then Glassmilk (5-10 ml) is added, mixedwell and left to stand for 10 min at ambient temperature. The glassmilkis spun down and washed 3 times with NEW WASH (0.5 ml) from the kit. Thewash buffer is removed from the Glassmilk which is to dry in air. TheDNA is eluted by incubating the dried Glassmilk with water (5-10 ml) at55° C. for 5-10 min. The aqueous supernatant containing the eluted DNAis recovered by centrifugation. The elution step can be repeated andsupernatants pooled;

[0128] Competent E. coli DH5α cells were obtained from Life TechnologiesLtd (MAX efficiency DH5α competent cells);

[0129] Mini-preparations of double stranded plasmid DNA were made usingthe RPM™ DNA preparation kit from Bio101 Inc. (cat. No 2070-400) or asimilar product—the kit contains alkaline lysis solution to liberateplasmid DNA from bacterial cells and glassmilk in a spinfilter to adsorbliberated DNA which is then eluted with sterile water or 10 mM Tris-HCl,1 mM EDTA, pH 7.5;

[0130] Serum free medium is OPTIMEM™ I Reduced Serum Medium, GibcoBRLCat. No. 31985;

[0131] LIPOFECTIN™ Reagent (GibcoBRL Cat. No. 18292-011) is a 1:1 (w/w)liposome formulation of the cationic lipidN-[1-(2,3-dioleyloxy)propyl]-n,n,n-trimethylammonium chloride (DOTMA)and dioleoyl phosphatidylethanolamine (DOPE) in membrane filtered water.It binds spontaneously with DNA to form a lipid-DNA complex—see Felgneret al. in Proc. Natl. Acad. Sci. USA (1987) 84, 7431;

[0132] G418 (sulphate) is GENETICIN™, GibcoBRL Cat No 11811, anaminoglycoside antibiotic related to gentamicin used as a selectingagent in molecular genetic experiments;

[0133] AMPLITAQ™, available from Perkin-Elmer Cetus, is used as thesource of thermostable DNA polymerase; and

[0134] General molecular biology procedures can be followed from any ofthe methods described in “Molecular Cloning-A Laboratory Manual” SecondEdition, Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory,1989).

EXAMPLE 1 Discovery and Establishment of Hybridoma Cell Line 806.077

[0135] BALB/C mice, 8 to 10 weeks old, were immunised subcutaneouslywith a primary dose of CEA (10 μg) in phosphate buffered saline solution(0.1 ml) and Freund's Complete adjuvant (0.1 ml). Two weeks later andagain 2 weeks later the animals were boosted with further doses of CEA(10 μg) in phosphate buffered saline (0.1 ml) mixed with Freund'sIncomplete adjuvant (0.1 ml). Thirty two weeks later the animals weregiven a final intravenous immunisation of CEA (10 μg) in phosphatebuffered saline and sacrificed three days later. The spleens wereremoved and prepared and fused with NS0 cells (available from theEuropean Collection of Animal Cell Cultures under the accession No.85110503) by standard methods (Kohler and Milstein, Nature (1975) 256,495). The resulting cells were distributed into 96-well culture dishesand incubated for 2 weeks. The supernatants from the resultinghybridomas were screened by EIA (enzyme immunoassay). From a total of1,824 wells generated from 5 fusions, 102 wells were positive againstnative CEA. In fusion 806, seventeen wells were found to be positive.The cells contained in these wells were cloned by limiting dilution, andthe resulting clones tested by EIA. Lines from 10/17 original wellscloned successfully. One line, designated 806.077, has been depositedwith the European Collection of Animal Cell Cultures under Accession No.96022936. The table below provides a summary of the antibody generationprogramme that led to discovery of the 806.077 antibody hybridoma.number number CEA* number rest number Wells +ve by finally Antigenhousing weeks fusions tested EIA selected Untreated normal  8-20 2813,920  99 0 CEA desialated normal  8-12 14 5,568  12* 0 CEA conjugatednormal 10-12  8 3,168  1 0 CEA Untreated isolator >30  5 1,824 102  3CEA

EXAMPLE 2 Preparation of 806.077 Antibody From Deposited Hybridoma CellLine ECACC No. 96022936

[0136] 2.1 Preparation from Serum Containing Medium

[0137] A 1 ml cryopreserved ampoule was removed from storage in liquidnitrogen and rapidly thawed in a 37° C. water bath. The contents wereaseptically transferred to a sterile 15 ml centrifuge tube. The cellswere resuspended by dropwise addition of 10 ml of Dulbecco's modifiedEagle's medium (DMEM) containing 10% (v/v) foetal calf serum (FCS)accompanied by gentle mixing. The suspension was centrifuged at 50×g for10 min, the supernatant aseptically removed and the pellet resuspendedin 5 ml of DMEM, 10% FCS and 1% L-glutamine in a 95% air 5% carbondioxide pre-gassed 25 ml tissue culture flask. The flask was incubatedat 36.5° C. in the dark.

[0138] After 3 days the flask was sub-cultured by passing the contentsof the entire flask into a larger 75 ml flask diluting with DMEM, 10%FCS and 1% L-glutamine (final viable density=2-3×10⁵ cells/ml). Furtherexpansion to 162 ml flasks was performed in a similar manner.

[0139] Culture supernatants for purification were prepared in 500 mlroller cultures in 850 ml roller bottles. Cultures were seeded at 2×10⁵viable cells/ml in pre-gassed roller bottles, rotated at 3 rpm andincubated at 36.5° C. Cultures were grown to maturity and harvestedtypically 500-800 hours after inoculation when the cell viability wasbelow 10% and IgG concentration had reached a maximum.

[0140] 2.2 Treatment of Culture Harvests

[0141] After harvest, roller bottle culture supernatants were clarifiedby centrifugation at 60×g for 30 minutes. Sodium azide (0.02% w/v) wasadded as a preservative to the clarified supernatant which was stored at4° C. in the dark until purification.

[0142] 2.3 Purification of 806.077 Antibody

[0143] 806.077 Antibody hybridoma supernatant (31) was adjusted to pH7.5 with dilute aqueous sodium hydroxide and filtered through a 0.45 μmfilter (Millipore MILLIDISK™). The filtered antibody supernatant wasloaded onto an affinity column of Protein G (for example Protein G FastFlow SEPHAROSE™, Pharmacia product code 17.0618.03; 5 cm i.d×6.5 cm=130ml;) equilibrated in phosphate buffered saline (“PBS”; 8 mM Na₂HPO₄, 1.5mM KH₂PO₄, 150 mM NaCl, 2.5 mM KCl, pH 7.3, for example as available intablet form for reconstitution from Oxoid) at 4° C. at a flow-rate of 4ml/min. The column was washed with PBS (260 ml) at the same flow rateand the antibody eluted with 100 mM sodium citrate pH 2.6, collectingfractions and monitoring the eluate by UV absorption (280 nm). The UVabsorbing fractions containing the antibody, were bulked, immediatelyadjusted to pH 7 and concentrated to about 2 mg/ml by ultrafiltrationusing a 30 kDa cut-off membrane (e.g. Amicon YM30). Dialysis, using a6-8 kDa porosity cut-off membrane (e.g. SPECTRAPOR™ 1) membrane, into 50mM tris-HCl pH 7.0 buffer yielded 110 mg 806.077 antibody, >95% pure bySDS-PAGE.

EXAMPLE 3 Selectivity of 806.077 Antibody

[0144] To assess selectivity, many human normal and tumour tissues havebeen screened for reactivity with the antibody 806.077, using sensitivethree-stage indirect immunohistology on acetone-fixed, frozen cryostatsections.

[0145] Immunohistology was carried out on sections of human tissueobtained either at resection surgery or at post mortem. To preserveoptimal morphology and antigenicity, tissues were obtained as fresh aspossible, cut into small pieces (about 0.5 cm³) and flash frozen inliquid nitrogen prior to storage at −80° C. Sections of tissue (6μ) werecut on a cryostat, mounted on polylysine coated slides (e.g. blueTECHMATE™ slides, Dako) and fixed in ice-cold acetone for 2 minutesbefore being wrapped in foil and stored at −80° C.

[0146] Slides were allowed to defrost at room temperature before beingunwrapped from the foil immediately prior to use. Each section wasoutlined with a diamond marker, and to each section was added either 100μl 806.077 antibody diluted to 2 μg/ml in Tris-buffered Saline (TBS), or100 μl CEA/NCA reactive control (A5B7 antibody) at 2 μg/ml in TBS, or100 μl MOPC isotype control (Sigma Chemical Company, St. Louis, U.S.A.,Cat. No. M 9269) at 2 μg/ml in TBS, or relevant positive control such asLP34 (Dako). All subsequent incubations were carried out at roomtemperature for 30 minutes in a humidified chamber: all wash steps werein TBS with 2 changes. After incubation, the slides were washed and 100μl of second antibody reagent, comprising {fraction (1/50)} rabbitanti-mouse immunoglobulins conjugated to horse radish peroxidase (DakoPatts) and ⅕ normal human serum (Sigma) in TBS was added to eachsection.

[0147] The slides were again incubated and washed in TBS. A finaldetecting antibody, 100 μl swine anti-rabbit immunoglobulin conjugatedto horse radish peroxidase ({fraction (1/50)} dilution with ⅕ normalhuman serum in TBS), was added to each section, incubated and washedthoroughly. DAB substrate (3,3′-diaminobenzidine tetrahydrochloride) wasprepared using 1 DAB tablet (Sigma) with hydrogen peroxide (17 μl) inTBS (17 ml), and added dropwise through a fast filter paper (e.g.Whatman Number 4). After 3 minutes incubation the excess DAB was tappedoff the slides and the slides were washed in TBS. After counter stainingwith haematoxylin (e.g. Mayer's Haematoxylin, Shandon) sections weredehydrated in alcohol and xylene, and mounted in non-aqueous syntheticmountant (e.g. E-Z mountant, Shandon) before examination under amicroscope.

[0148] The areas of antibody bonding were visualised by brown stainingon the section. A scoring system was used to evaluate the degree ofbinding of 806.077 antibody to tissues:

[0149] +++ (strong)=antibody binding to >75% tumour cells

[0150] ++ (moderate)=antibody binding to 50%-75% tumour cells

[0151] + (weak)=antibody binding to 25% -50% tumour cells

[0152] +/− (minimal)=non-focal antibody binding to a small area oftumour cells − =no staining

[0153] Carcinoembryonic antigen (CEA) is a member of the immunoglobulingene superfamily with one predicted variable-like domain region (Ndomain; 108 amino acids) and three sets of constant domain-like regionsA1B1, A2B2 and A3B3; 92 amino acids for A domains and 86 amino acids forB domains (Hefta, 1992, Cancer Research 52:5647-5655. In addition, CEApossesses two signal peptides, one at the amino terminus and one at thecarboxyl terminus. Both are removed during post-translationalprocessing, the one at the carboxy terminus being replaced by aglycosylphosphatidylinositol (GPI) moiety.

[0154] A large number of CEA-related proteins with varying homology toCEA have been reported (Thompson, 1991, J. of Clinical LaboratoryAnalysis, 5: 344-366). These include non-specific cross reactingantigens, NCA 1 and 2. These related proteins are expressed on a rangeof normal tissues including granulocytes and normal lung epithelium. Themajority of anti-CEA monoclonal antibodies generated so far, cross reactwith one of these related proteins and thus react with a range of normaltissues and often react strongly with either granulocytes or lungepithelium.

[0155] Anti-CEA antibody, 806.077 was identified as being CEA selective,exhibiting no cross reactivity to granulocytes and only minimal stainingto 4/14 normal lung tissues tested.

[0156] 806.077 antibody was initially screened for tumour and NCAselectivity as a tissue culture supernatant. The screens were carriedout using the supernatant neat and diluted at 1:10 and demonstratedequivalent binding of the antibody to, colon tumours when compared toA5B7, but much reduced binding to normal lung and spleen tissues whencompared to the same antibody. The antibody was affinity purified (asdescribed in Example 2) and the screens repeated and extended to includefurther tumours and tissue types.

[0157] The antibody was titrated against a panel of colo-rectal tumoursections and this screen demonstrated the optimum screeningconcentration of 806.077 antibody to be 2 μg/ml. All subsequent screenswere carried out using the antibody at this concentration. The resultsof these screens were as follows. The reactivity of 806.077 antibody wascompared against A5B7 (also screened at 2 μg/ml) against the followingtumours/normal tissues:

[0158] 806.077 antibody Tumour reactivity:

[0159] Colon tumours (n=17).

[0160] Moderate to strong reactivity (++/+++ equivalent to A5B7) wasseen to all 17 tumours tested.

[0161] Breast tumours (n=6).

[0162] Moderate/weak staining (+/++), 2/6 tumours; minimalstaining(+/−), 2/6 tumours.

[0163] NSCLC tumours (n=6).

[0164] Strong staining (+++), 2/6 tumours; moderate staining (++), 1/6and weak staining (+), 2/6 tumours.

[0165] Gastric tumours (n=2).

[0166] Strong staining (+++), 1/2 tumours; weak staining (+) 1/2tumours.

[0167] Ovary tumours (n=3) and prostate tumours (n=3).

[0168] No staining was seen to any of these tumours.

[0169] In all cases, equivalent reactivity was seen with A5B7.

[0170] Normal tissue reactivity:

[0171] Lung (NCA reactivity) (n=14).

[0172] Weak staining (+), 4/14 lung tissues; no staining (−) 10/14tissues.

[0173] A5B7 bound moderately (++), 1/14 lung tissues; weakly (+), 10/14tissues and minimally (+/−), 1/14 tissues.

[0174] Spleen (granulocyte/NCA reactivity) (n=6).

[0175] No staining was seen to any of the spleen tissues tested.

[0176] A5B7 bound moderately (++), 1/6 tissues and weakly (+), 5/6tissues

[0177] Post mortem normal tissues (n=13).

[0178] Moderate/weak reactivity (++/+) was seen only to oesophagus,skin, colon and pancreas tissues (CEA expressing normal tissues).Similar binding was seen with A5B7. In addition to the positive tissues,colon, skin, oesophagus and pancreas, the negative tissues were:cerebellum, mid-brain, cerebrum, smooth muscle, liver, kidney, aorta,stomach, heart.

EXAMPLE 4 Generation of 806.077 Antibody F(ab′) Fragment

[0179] Ficin (10 mg) was suspended in a solution of 50 mM cysteine (3ml; BDH 37218) and 50 mM tris-HCl pH 7.0 and incubated at 37° C. for 30minutes. Excess cysteine was removed by size exclusion chromatography(Sephadex™ G-25 column, 1.5 cm×25 cm; Pharmacia) in 50 mM tris-HCl pH7.0 buffer. The reduced ficin concentration was determined by monitoringUV absorbance at A280 nm (assuming that a 1 mg/ml solution has anabsorbance reading of 2 in a 1 cm cell) and was found to be 1.65 mg/ml.

[0180] A solution of 806.077 antibody (100 mg) in 50 mM tris-HCl bufferpH 7.0 (50 ml) and freshly reduced ficin (5 mg; 3 ml of the abovesolution) was digested at 37° C. over 20 hours. The digest was thendiluted with an equal volume of PBS and loaded onto a Protein G affinitycolumn (Pharmacia SEPHAROSE™ Fast Flow, 5.0 cm i.d×6.5 cm=125 ml;previously equilibrated with 50 mM tris-HCl pH 7.0 buffer at 4° C.), ata constant flow-rate of 3 ml/min. The column was washed with 50 mMsodium acetate pH 4.0 (250 ml) to remove low M.W. fragments, followed by50 mM sodium citrate pH 2.8 to elute the F(ab′)₂, monitoring the UVadsorbance of the eluate at A280 nm. The F(ab′)₂ containing eluate wasadjusted to pH 7 and buffer exchanged into 100 mM sodium phosphate/100mM sodium chloride/1 mM EDTA pH 7.2 by dialysis and concentrated to 8mg/ml by membrane filtration using a 10 kDa cut-off (e.g. Amicon™ YM10)assuming that a 1 mg/ml solution has an absorbance reading at 280 nm of1.4 in a 1 cm cell. A 65% yield of 42 mg 95% pure F(ab′)₂ was obtained.

EXAMPLE 5 Preparation of 806.077 Antibody F(ab′)₂-Carboxypeptidase G2Conjugate

[0181] The linker for 806.077 antibody F(ab′)₂ derivatisation was SATA(S-acetyl thioglycollic acid N-hydroxysuccinimide ester, Sigma, productcode A 9043) The linker for carboxypeptidase G2 (CPG2) derivatisationwas SMPB [4-(p-maleimidophenyl)butyric acid N-hydroxysuccinimide ester,Sigma, product code M6139]

[0182] 5.1 F(ab′)₂ Derivatisation:

[0183] To a solution of the F(ab′)₂ fragment (40 mg, prepared asdescribed in Example 4) in 100 mM phosphate/100 mM NaCl/1 mM EDTA pH 7.2(buffer A; 5 ml) was mixed with SATA (0.28 mg) in DMSO (28 μl). After 40minutes at room temperature the resulting solution was applied to adesalting column (SEPHADEX™ G-25, 1.5 cm i.d×50 cm=100 ml; equilibratedin buffer A at 4° C.) at a flow-rate of 1.2 ml/min. to remove excessreagents. The eluate was monitored by UV absorption at A280 nm. The SATAderivatised F(ab′)₂ was pooled and mixed with 10% v/v 500 mMhydroxylamine HCl/500 mM sodium phosphate/30 mM EDTA pH 8.0 for 60minutes at room temperature to deacetylate the derivatised F(ab′)₂. Theprotein concentration was determined by UV absorption at 280 nm assumingthat a 1 mg/ml solution has an absorbance reading of 1.4 in a 1 cm cell.The solution was diluted to about 1 mg/ml with buffer A. The linkerloading was determined by Ellman's-SH assay and found to be 1.8-2.0linkers/mole F(ab′)₂.

[0184] 5.2 CPG2 Derivatisation:

[0185] Large scale purification of CPG2 from Pseudomonas RS-16 wasdescribed in Sherwood et al. (1985), Eur, J. Biochem., 148, 447-453.Preparation of F(ab′)₂ and IgG antibodies coupled to CPG enzyme may beeffected by known means and has been described for example in PCT WO89/10140. CPG may be obtained from Centre for Applied Microbiology andResearch, Porton Down, Salisbury, Wiltshire SP4 0JG, United Kingdom.CPG2 may also be obtained by recombinant techniques. The nucleotidecoding sequence for CPG2 has been published by Minton, N. P. et al.,Gene, (1984) 31, 31-38. Expression of the coding sequence has beenreported in E. coli (Chambers. S. P. et al., Appl. Microbiol,Biotechnol. (1988), 29, 572-578) and in Saccharomyces cerevisiae(Clarke, L. E. et al., J. Gen Microbiol, (1985) 131, 897-904). Totalgene synthesis has been described by M. Edwards in Am. Biotech. Lab(1987), 5, 38-44. Expression of heterologous proteins in E. coli hasbeen reviewed by F. A. O. Marston in DNA Cloning Vol. III, PracticalApproach Series, IRL Press (Editor D M Glover), 1987, 59-88. Expressionof proteins in yeast has been reviewed in Methods in Enzymology Volume194, Academic Press 1991, Edited by C. Guthrie and G R Fink.

[0186] CPG enzyme is available from Sigma Chemical Company, Fancy Road,Poole, Dorset, U.K. CPG enzyme was described in: Goldman, P. and Levy,C. C., PNAS USA, 58: 1299-1306 (1967) and in: Levy, C. C. and GoldmanP., J. Biol. Chem., 242: 2933-2938 (1967). Carboxypeptidase G3 enzymehas been described in Yasuda, N. et al., Biosci. Biotech. Biochem., 56:1536-1540 (1992). Carboxypeptidase G2 enzyme has been described inEuropean Patent 121 352.

[0187] CPG2 (50 mg; recombinant enzyme from E. coli) was dialysed into100 mM sodium phosphate/100 mM sodium chloride pH 7.2 (=buffer B) anddiluted to 8 mg/ml, assuming that a 1 mg/ml solution has an absorbancereading at 280 nm of 0.6 in a 1 cm cell.

[0188] SMPB(Sigma) was dissolved in DMSO at 10 mg/ml. CPG2 (50 mg inbuffer B at 8 mg/ml) was mixed with the SMPB solution (0.108 ml; 1.08mg), and reacted at room temperature for 120 minutes. Excess reagentswere removed on a desalting column (Sephadex G-25, 1.5cm i.d×50 cm=100ml; equilibrated in buffer B at 4° C.) at 1.2 ml/min. Derivatised CPG2was pooled and the concentration determined by UV A280 nm, assuming thata 1 mg/ml solution has an absorbance reading at 280 nm of 0.6 in a 1 cmcell. The solution was diluted to a CPG2 concentration of about 1 mg/ml.The linker loading was determined by a ‘reverse’ Ellman's assay, byadding a known amount of 2-mercaptoethanol to the maleimido-derivatisedCPG2 and assaying unreacted -SH. A linker loading of 2.0-2.4linkers/mole CPG2 was found.

[0189] 5.3 Conjugation:

[0190] Equal weights of the deacetylated derivatised F(ab′)₂ andderivatised CPG2 were mixed under nitrogen and the mixture (about 80 ml,at a total protein concentration of about 1 mg/ml) left at roomtemperature for 20 h. The reaction was terminated by the addition of 10%v/v 100 mM aqueous glycine. The crude conjugation mixture was bufferexchanged by dialysis into a low salt buffer (50 mM sodium acetate pH6.0) and applied to a dye-ligand affinity column (where the dye binds toCPG2 e.g. ACL Mimetic Green 1, 2.5cm i.d×10 cm=50 ml) at 4° C.equilibrated in the same buffer, to remove unreacted derivatisedF(ab′)₂. The conjugate and derivatised CPG2 were eluted with 50 mMacetate/500 mM NaCl pH 6.0, at a flow rate of 2.0 ml/min monitoring theelution by UV (A280 nm).

[0191] The crude conjugate, still containing derivatised CPG2, wasconcentrated using a 10 kDa cut-off ultrafiltration device (e.g. AmiconYM10™) to about 12 ml, at 5 mg/ml total protein concentration and 10%v/v 10 mM zinc sulphate (Sigma Z 0251) in water was added to replenishzinc lost to the CPG2 in the process. Further chromatography by sizeexclusion (e.g. SEPHACRYL S-300HR™ Pharmacia, 2.5 cm i.d×25 cm=500 ml)at 4° C. in 50 mM sodium acetate/150 mM sodium chloride pH 6.0 at aflow-rate of 1 ml/min., collecting fractions and monitoring by UV A280nm, resulted in the fractionation of the conjugate and its separationfrom unreacted derivatised CPG2, as determined by SDS-PAGE of columnfractions.

[0192] The peak containing conjugate (with ratios of F(ab′)₂:CPG2 of1:2, 1:1 and 2:1) was pooled and concentrated by ultrafiltration to 1.3mg/ml, the protein concentration being determined by monitoring UVadsorbance at A280 nm (assuming 1 mg/ml has an absorbance of 1.0).Purity of the conjugate was determined by SDS PAGE and found to containa total of 12 mg conjugate with the composition 65% 1:1 ratio conjugate,20% 1:2 or 2:1 ratio conjugate with <5% free derivatised F(ab′)2 and <5%free derivatised CPG2.

EXAMPLE 6 Anti-tumour Activity of 806.077 Antibody F(ab′)₂-CPG2Conjugate in Combination With A Prodrug

[0193] The anti-tumour activity of the 806.077 antibody F(ab′)₂-CPG2conjugate prepared as described in Example 5 was evaluated incombination with the prodrugN(4-[N,N-bis(2-chloroethyl)amino]-phenoxycarbonyl)-L-glutamic acid(called “PGP” in this example, is described in Example 1 in U.S. Pat.No. 5,405,990 and Blakey et al., Br. J. Cancer 72, 1083-88, 1995) in ahuman colorectal tumour xenograft model.

[0194] Groups of 8-10 female athymic nude mice were injected s.c. with1×10⁷ LoVo colorectal tumour cells (ECACC no 87060101). When the tumourswere 4-5 mm in diameter either 806.077 antibody F(ab′)₂-CPG2 conjugate(250 U CPG2 enzyme activity Kg⁻¹) or phosphate buffered saline (170 mMNaCl, 3.4 mM KCl, 12 mM Na₂HPO₄, 1.8 mM KH₂PO₄, pH 7.2) was injectedintravenously (i.v). Seventy-two hours later PGP prodrug was injectedi.p. (3 doses of 40 mg/Kg at 1 h intervals). The length of the tumoursin two directions was then measured three times a week and the tumourvolume calculated using the formula:

Volume=Π/6×D ² ×d

[0195] where D is the larger diameter and d is the smaller diameter ofthe tumour. Tumour volume was expressed relative to the tumour volume atthe time of initiation of the prodrug arm of the therapy. Theanti-tumour activity was compared with control groups given PBS insteadof either conjugate or prodrug. Anti-tumour activity was expressed bothas a growth delay defined as the time it takes treated tumours toincrease their volume by 4-fold minus the time it takes control tumoursto increase their volume 4-fold and as a T/C value defined as the volumeof the treated tumour divided by the volume of the control tumour 14days after prodrug administration. Statistical significance of theanti-tumour effects was judged using the analysis of variance (one-way)test.

[0196] The anti-tumour activity of 806.077 antibody F(ab′)₂-CPG2conjugate in combination with PGP prodrug are shown in FIG. 1 and theanti-tumour data is summarised below.

[0197] Anti-Tumour Activity of 806.077 Antibody F(ab′)₂-CPG2 Conjugatein Combination With PGP Prodrug in LoVo Tumour Xenografts Dose T/CGrowth delay Significance Conjugate (U/kg) (%) (days) (p) 806.077F(ab′)₂-CPG2 250 16.5 14 <0.01 500 4.7 22 <0.01

[0198] The results demonstrate that the 806.077 antibody F(ab′)₂-CPG2conjugate in combination with the PGP prodrug produce tumour regressionsand prolonged growth delays which were statistically significantcompared with control groups.

EXAMPLE 7 Cloning and Sequencing of the Variable Regions of 806.077Antibody Heavy and Light Chain Genes

[0199] 7.1 Preparation of Cytoplasmic RNA

[0200] There are several procedures for the isolation of polyA+ mRNAfrom eukaryotic cells (Sambrook J., Fritsch E. F., Maniatis T.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Second Edition, 1989, Chapter 8, p3 herein referred to as“Maniatis”). In this particular case cytoplasmic RNA was prepared asdescribed by Favoloro et al., Methods in Enzymology 65, 718-749, from afrozen hybridoma cell pellet containing 1×10⁹ cells which had beenstored at −80° C.

[0201] The cells were resuspended in 5 ml ice-cold lysis buffer (140 mMNaCl, 1.5 mM MgCl₂, 10 mM Tris-HCl pH 8.6 and 0.5% NP40 (a polyglycolether nonionic detergent; Nonylphenoxy Polyethoxy Ethanol, Sigma Cat.No. 127087-87-0)) containing 400 u of a ribonuclease inhibitor(RNAguard; Pharmacia Cat. No. 27-0815-01) and vortexed for 10 s. Thissolution was overlayed on an equal volume of ice cold lysis buffercontaining 24% (w/v) sucrose and 1% NP-40 and stored on ice for 5 min.The preparation was then centrifuged at 4000 rpm for 30 min at 4° C. ina bench top centrifuge (Sorval RT6000B ) after which, the uppercytoplasmic phase was removed to an equal volume of 2×PK buffer (200 mMTris (pH7.5), 25 mM EDTA, 300 mM NaCl and 2% (w/v) SDS). Proteinase K(Sigma, Cat No. P2308) was added to a final concentration of 200 μg/mland the mixture incubated at 37° C. for 30 min.

[0202] The preparation was extracted with an equal volume ofphenol/chloroform, the aqueous phase removed and 2.5 vol ethanol addedand mixed. This solution was then stored at −20° C. overnight. RNA wascollected by centrifugation (4000 rpm, 30 min at 4° C. in a bench topcentrifuge, Sorval RT6000B), the supernatant decanted and the pelletdried in a vacuum dessicator after which it was dissolved in 250 μldiethylpyrocarbonate (DEPC)-treated water (prepared as described inManiatis, referenced above). The RNA content was measured byspectrophotometry and the concentration calculated assuming anabsorbance at 260 nm of 1=40 μg/ml.

[0203] 7.2 Preparation of First Strand Variable Region cDNA

[0204] A number of methods for the synthesis of cDNA are reviewed inManiatis (Chapter 8). The oligonucleotide primers used were mainly basedon those proposed by Marks et al. J. Mol. Biol (1991) 222, 581-597. ThecDNA in this case was prepared as described below. RNA (5 mg) was mixedin a microcentrifuge tube with 10 μl 5× reverse transcriptase buffer[250 mM Tris (pH8.3), 40 mM MgCl₂ and 50 mM DTT], 1 μl forward primer(25 pM), 10 μl 1.25 mM dNTPs, 5 μl 10 mM DTT, 0.5 μl RNAguard(Pharmacia) to which DEPC-treated H2O was added to obtain a volume of 50μl. The reaction mix was heated to 70° C. for 10 min and then cooledslowly to 37° C., after which 100μ (0.5 μl) M-MLV reverse transcriptase(Pharmacia Cat. No. 27-0925-01) were added and the reaction incubated at37° C. for 1 h. The forward primer used for the generation of the lightchain cDNA was oligonucleotide CK2FOR (SEQ ID NO: 1) which is designedto hybridise to the CK constant region of murine kappa light chaingenes. For the heavy chain cDNA the forward primer CG1FOR (SEQ ID NO: 2)was used which hybridises to the CH1 constant domain of murine IgG1.

[0205] 7.3 Amino Acid Sequencing

[0206] The heavy and light chains of the 806.077 antibodies wereisolated by SDS-PAGE and Western blotting and submitted for N-terminalamino acid sequencing. The results showed that the N-terminus of thelight chain was chemically blocked, however, sequence data was obtainedfor the first 34 N-terminal residues of the heavy chain (SEQ ID NO: 3).On the basis of this amino acid sequence a specific DNA back primer wasdesigned for 806.077 heavy chain variable region PCR. This primer wascalled SP1back (SEQ ID NO: 7).

[0207] 7.4 Isolation of Antibody Gene Fragments By PCR

[0208] Isolation of 806.077 heavy and light chain variable region geneswas performed using the cDNA prepared as described above as template.General reaction conditions were as follows.

[0209] To 5 μl of the cDNA reaction was added 5 μl dNTPs (2.5 mM), 5 μl10× Enzyme buffer (500 mM KCl, 100 mM Tris (pH8.3), 15 mM MgCl₂ and 0.1%gelatin), 1 μl of 25 pM/μl back primer, 1 μl of 25 pM/μl forward primer,0.5 μl thermostable DNA polymerase and DEPC-treated water to obtain avolume of 50 μl . The PCR conditions were set for 25 cycles at 94° C.for 90 s; 55° C. for 60 s; 72° C. for 120 s, ending the last cycle witha further 72° C. for 10 min incubation.

[0210] Using the general reaction conditions, the forward primer usedfor the generation of the light chain cDNA was oligonucleotide CK2FOR(SEQ ID NO: 1) and the for the heavy chain cDNA oligonucleotide CG1FOR(SEQ ID NO: 2). A number of reactions using a variety of different backprimers were performed for both the heavy and light chains to obtaindesired specific PCR products.

[0211] In the case of the 806.077 light chain, on analysis a specificPCR product was obtained using the back primers VK1 back (SEQ ID NO: 4)and VK4back (SEQ ID NO: 5). Similarly specific PCR products wereobtained for the heavy chain using VH1back (SEQ ID NO: 6) and SP1backprimers (SEQ ID NO: 7). Reaction products were analysed on a 2% agarosegel. Products of the expected size, were excised and the DNA purified.

[0212] 7.5 Cloning of the PCR Products into Bluescript KS+ Vector

[0213] For each antibody fragment, both the 5′ region (back primer)oligonucleotide and the 3′ region (forward primers) introduced arestriction site. The discrete PCR products were for both the VH and VKPCR reactions were therefore able to be cloned into the Bluescriptvector KS+ (Stratagene Cloning Systems) via the appropriate enzymerestriction sites using standard DNA manipulation methods (e.g. PCRproducts VH1back/CG1For was cloned via PstI/HindIII and VK4back/CK2Forvia SacI/HindIII). DNA was prepared from the clones obtained andrigorous sequencing of at least 12 clones of each construct performedusing automated fluorescent sequencing equipment (Applied Biosystems).The sequences were reviewed, compared and aligned using suitablecomputer software. Consensus sequences for both the VH and VK genes wereobtained and subsequently translated to their corresponding amino acidsequence.

[0214] The DNA and amino acid sequences obtained for the 806.077 lightchain variable (VK) region are described in SEQ ID NO: 8 and SEQ ID NO:9 respectively. The DNA and amino acid sequences obtained for the806.077 heavy chain variable (VH) region are described in SEQ ID NO: 10and SEQ ID NO: 11 respectively. A clone containing the light chain wasdesignated VK4, and a clone containing the heavy chain sequnece wasdesignated VH14A.

EXAMPLE 8 Construction of Chimaeric Light Chain and Heavy Chain Fd Genes

[0215] The heavy and light chain genes which had been cloned intoBluescript (VK4 and VH14A in Example 7) were isolated by PCR usingprimers which allowed specific amplification of only the variable regionthe appropriate genes but also introduced new unique enzyme restrictionsites. These restriction sites enabled the variable region genefragments to be cloned in frame with DNA fragments coding for both theappropriate antibody signal sequences and human constant regions. Thesignal and constant region sequences for the light and heavy chain Fdhad each been previously cloned into pNG3 and pNG4, derivatives of thepSG5 Eukaryotic plasmid expression vector.

[0216] The vector pNG3 was prepared as follows. Plasmid pSG5(Stratagene, Cat. No. 216201) was digested with SalI and XbaI to removethe existing SV40 promoter and polylinker sequence. A new polylinker wasintroduced by use of oligonucleotides SEQ NOS: 34 and 35 which werehybridised and cloned into the SalI and XbaI cut pSG5 plasmid to giveplasmid pNG1. The pNG1 plasmid was cut with BglII and HindIII and theBglII-HindIII CMV promoter fragment from pcDNA3 (Invitrogen, Cat. No.V790-20) cloned into this site to give plasmid pNG2. Finally, the polyAregion from pSG5 was isolated by PCR as described in Example 7, section7.4 but using oligonucleotide sequences SEQ ID NOS: 36 and 37 withplasmid pSG5. The PCR product was cut with XmaI and BamHI, purified byelectrophoresis on a 2% agarose gel, isolated (e.g. with GENECLEAN, seeexample 7) then ligated into the Xmal-BamHI cut pNG2 plasmid to givepNG3.

[0217] The pNG4 vector was prepared as follows. The pNG3 vector wasfurther modified such hat the SacI restriction enzyme recognition sitein the cloned CMV promoter fragment was corrupted by changing the DNAsequence. This was achieved by the use of a two step PCR mutagenesisreaction using the pNG3 vector as a template. The PCR used-twocomplementary oligonucleotide primers (SEQ ID NOS: 38 and 39) to mutatethe Sac I recognition sequence and 2 flanking primers (SEQ ID NOS: 40and 41) for product amplification. Two Primer pairs (SEQ ID NOS: 38 and41) and (SEQ ID NOS: 39 and 40) were used in a standard PCR reaction (asdescribed in Example 7, section 7.4) to obtain the initial 2 PCRproducts, which were isolated by electrophoresis on 2% agarose gels.Equimolar amounts of each product were mixed and reamplified using theflanking primers (SEQ ID NOS: 40 and 41) under the standard PCR reactionconditions to splice together and amplify the final PCR product. Thisproduct was subsequently digested with the restriction enzymes NcoI andHindIII and cloned into the appropriately restricted and prepared pNG3vector such that the mutated (SacI site minus) fragment replaced theoriginal pNG3 NcoI-Hind III (SacI site plus) fragment. This new vectorwas named pNG4.

[0218] A clone of the 806.077 murine light chain in the Bluescript KS+vector (VK4) was taken and amplified using the oligonucleotide primers077VK-UP (SEQ ID NO: 12) and 077VK-DOWN (SEQ ID NO: 13). Similarly a806.077 heavy chain clone (VH14A) was amplified using 077VH-UP (SEQ IDNO: 14) and 077VH-DOWN (SEQ ID NO:15). The PCR was performed as follows:To 100 ng of plasmid DNA was added 5 μl dNTPs (2.5 mM), 5 μl 10× Enzymebuffer (see above), 1 μl of 25 pM/μl back primer, 1 μl of 25 pM/μlforward primer, 0.5 μl thermostable DNA polymerase and DEPC-treatedwater to obtain a volume of 50 μl. The PCR conditions were set for 15cycles at 94° C. for 90 s; 55° C. for 60 s; 72° C. for 120 s, ending thelast cycle with a further 72° C. for 10 min incubation. The productswere analysed on a 2% agarose gel. The DNA was purified and the DNAfragment digested with the relevant restriction enymes in preparationfor subsequent vector cloning.

[0219] For secretion of antibody light chain, a double stranded DNAcassette which contained both the information for a Kozak recognitionsequence and a light chain signal sequence was designed. The cassetteconsisted of two individual oligonucleotides (SEQ ID NOS: 42 and 43)which were hybridised and subsequently cloned between cloned between theHindIII and SacII restriction site of the pNG3 plasmid (which had beenappropriately restricted and isolated using standard methodology) tocreate the vector pNG3-Vkss. The DNA sequence of SEQ ID NO: 46, whichcontains the sequence for the human light chain kappa constant region,was digested with XmaI and XhoI and inserted between the XhoI and Xmalcut pNG-Vkss plasmid to give the vector pNG3-Vkss-HuCk (NCIMB no.40798). Furthermore, a neomycin resistance gene expression cassette wascloned into pNG3-Vkss-HuCk (from the pSG5 plasmid variant pSG5-Neovector, supplied from S. Green, Zeneca Pharmaceuticals; alternativesources include vectors such as pMC1neo, Stratagene cat. no. 213201).The neomycin resistance gene expression cassette was cloned as an XbaIfragment and cloned into the XbaI site of the pNG3-Vkss-HuCk and theorientation was checked using restriction enzyme digestion. This gaverise to the plasmid pNG3-Vkss-HuCk-Neo (NCIMB 40799). The light chaingene sequence described above was inserted, in frame, by cloningdirectly between the SacII and XhoI sites of the pNG3-Vkss HuCk-neovector. The PCR fragment obtained for the light chain gene was digestedwith SacII and XhoI restriction enzymes and cloned into the similarlyrestricted expression vector containing the VK signal and HuCK constantregion coding sequences. The chimaeric 806.077 light chain sequencecreated is shown in SEQ ID NOS: 16 and 17.

[0220] Similarly, for secretion of antibody heavy chain, a doublestranded DNA cassette which contained both the information for a Kozakrecognition sequence and a heavy chain signal sequence was designed. Thecassette consisted of two individual oligonucleotides (SEQ ID NOS: 44and 45) which were hybridised and subsequently cloned between clonedbetween the HindIII and EcoRI restriction site of the pNG4 plasmid(which had been appropriately restricted and isolated using standardmethodology) to create the vector pNG4-VHss. Heavy chain gene sequencescould thus be inserted in frame, by cloning directly between the EcoRIand SacI sites of the pNG4-VHss vector. The DNA sequence of SEQ ID NO:47, which contains the coding sequence for human heavy chain IgG2CH1′constant region (SEQ ID NOS: 22 and 23) was digested with SacI and XmaIand cloned into pNG4-VHss cut with SacI and XmaI to give the vectorpNG4-VHss-HuIgG2CH1′ (NCIMB no. 40797). The PCR fragment obtained forthe heavy chain gene was digested with EcoRI and SacI restrictionenzymes and cloned into the similarly restricted expression vectorpNG4-VHss-HuIgG2CH1′ containing the VH signal and HuIgG2 CH1′ constantregion coding sequences. The chimaeric 806.077 HuIgG2 Fd chain sequencecreated is shown in SEQ ID NOS: 18and 19.

[0221] In some instances it may be preferable to use other classes ofchimaeric heavy chain Fd constructs. To this end, variants of the heavychain vector are made containing HuIgG1CH1′ (SEQ ID NOS: 20 and 21) orHuIgG3CH1′ (SEQ ID NOS: 24 and 25) which are substituted for theHuIgG2CH1′ (SEQ ID NOS: 22 and 23) gene.

[0222] The sequences shown in SEQ ID NOS: 46 and 47 are prepared by avariety of methods including those described by Edwards (1987) Am.Biotech. Lab. 5, 38-44, Jayaraman et al. (1991) Proc. Natl. Acad. Sci.USA 88, 4084-4088, Foguet and Lubbert (1992) Biotechniques 13, 674-675and Pierce (1994) Biotechniques 16, 708. Preferably, the sequences shownin SEQ ID NOS: 46 and 47 are prepared by a PCR method similar to thatdescribed by Jayaraman et al. (1991) Proc. Natl. Acad. Sci. USA 88,4084-4088.

[0223] Once the individual heavy and light chain sequences wereconstructed a heavy chain Fd gene expression cassette (including bothpromoter and gene was excised as a BglII/SalI fragment and clonedbetween into the BamHI/SalI sites of the light chain vector to produce aco-expression vector construct. This construct was transfected into NS0myeloma cells (ECACC No. 85110503) via standard techniques ofelectroporation and transfectants selected for the property of G418resistance, a trait which is carried as a selectable marker on theexpression plasmid construct.

[0224] Alternatively the complete heavy chain Fd and light chain genesmay simply be excised from their respective vectors as HindIII/XmaIfragments and subsequently cloned into other expression vector systemsof choice.

EXAMPLE 9 Hybridization Test of Nucleic Acid Variations of SpecificNucleic Acid Sequences

[0225] 9.1 Hybridisation Test

[0226] A method for detecting variant nucleic acids containing sequencesrelated to specific 806.077 antibody sequences is described. Thesevariant nucleic acids may be present in a variety of forms such as theDNA from bacterial colonies or the DNA/RNA from eukaryotic cells fixedon to a membrane as described above in the screening of a cDNA libraryor as fragments of purified nucleic acid separated by gelelectrophoresis and then transfered to a suitable membrane as for thetechniques of Northern (Maniatis et al, Chapter 7, p39) or Southern(Maniatis, chapter 9, p31) hybridisation.

[0227] 9.2 Hybridisation Probe

[0228] Hybridisation probes may be generated from any fragment of DNA orRNA encoding the specific 806.077 antibody nucleic sequence of interest,more specifically from the variable region, particularly the regionencoding CDR3 of this region. A synthetic oligonucleotide or itscomplementary sequence can be used as a specific probe for the CDR3encoding region.

[0229] A hybridisation probe can be generated from a syntheticoligonucleotide by addition of a radioactive 5′ phospate group from[γ-³²P]ATP by the action of T4 polynucleotide kinase. 20 pmoles of theoligonucleotide are added to a 20 μl reaction containing 100 mM Tris,pH7.5, 10 mM MgCl₂, 0.1 mM spermidine, 20 mM dithiothreitol (DOT), 7.55μM ATP, 55 μCi [γ-³²P]ATP and 2.5u T4 polynucleotide kinase (PharmaciaBiotechnology Ltd, Uppsala, Sweden). The reaction is incubated for 30minutes at 37° C. and then for 10 minutes at 70° C. prior to use inhybridisation. Methods for the generation of hybridisation probes fromoligonucleotides (chapter 11) or from DNA and RNA fragments (chapter 10)are given in Maniatis. A number of proprietary kits are also availablefor these procedures.

[0230] 9.3 Hybridisation Conditions

[0231] Filters containing the nucleic acid are pre-hybridised in 100 mlof a solution containing 6× SSC, 0.1% SDS and 0.25% dried skimmed milk(Marvel™) at 65° C. for a minimum of 1 hour in a suitable enclosedvessel. A proprietary hybridisation apparatus such as model HB-1 (TechneLtd) provides reproducible conditions for the experiment.

[0232] The pre-hybridisation solution is then replaced by 10 ml of aprobe solution containing 6×SSC, 0.1% SDS, 0.25% dried skimmed milk(e.g. Marvel™) and the oligonucleotide probe generated above. Thefilters are incubated in this solution for 5 minutes at 65° C. beforeallowing the temperature to fall gradually to below 30° C. The probesolution is then discarded and the filters washed in 100 ml 6×SSC, 0.1%SDS at room temperature for 5 minutes. Further washes are then made infresh batches of the same solution at 30° C. and then in 10° C.increments up to 60° C. for 5 minutes per wash.

[0233] After washing, the filters are dried and used to expose an X-rayfilm such as Hyperfilm™ MP (Amersham International) at −70° C. in alight-tight film cassette using a fast tungstate intensifying screen toenhance the photographic image. The film is exposed for a suitableperiod (normally overnight) before developing to reveal the photographicimage of the radio-active areas on the filters. Related nucleic acidsequences are identified by the presence of a photographic imagecompared to totally unrelated sequences which should not produce animage. Generally, related sequences will appear positive at the highestwash temperature (60° C.). However, related sequences may only showpositive at the lower wash temperatures (50, 40 or 30° C.).

[0234] These results will also depend upon the nature of the probe used.Longer nucleic acid fragment probes will need to be hybridised forlonger periods at high temperature but may remain bound to relatedsequences at higher wash temperatures and/or at lower saltconcentrations. Shorter, mixed or degenerate oligonucleotide probes mayrequire less stringent washing conditions such as lower temperaturesand/or higher Na⁺ concentrations. A discussion of the considerations forhybridisation protocols is provided in Maniatis (chapter 11).

EXAMPLE 10 Pharmaceutical Compositions

[0235] The following illustrates representative pharmaceutical dosageforms containing 806.077 antibody which may be used for therapy incombination with a suitable prodrug.

[0236] Injectable Solution for ADEPT

[0237] A sterile aqueous solution, for injection, containing per ml ofsolution: 806.077 antibody - CPG2 conjugate  1.0 mg Sodium acetatetrihydrate  6.8 mg Sodium chloride  7.2 mg Tween 20 0.05 mg

[0238] A typical dose of conjugate for adult humans is 30 mg followed 3days later by three 1 g doses of prodrug administered at hourlyintervals. Suitable CPG2 conjugates are any one of those conjugatesdescribed in Examples 105 and 106. Conjugates with HCPB may replace theCPG2 conjugate in the table. Suitable HCPB conjugates are any one ofthose conjugates described in Examples 48-101.

[0239] Injectable Solution for Tumour Immunotherapy

[0240] A sterile aqueous solution, for injection, containing per ml ofsolution: 806.077 antibody - B7 conjugate  1.0 mg Sodium acetatetrihydrate  6.8 mg Sodium chloride  7.2 mg Tween 20 0.05 mg

[0241] A typical dose of conjugate for adult humans is 30 mg. A suitableconjugate is described in Example 104.

EXAMPLE 11 Construction of Initial 806.077 Humanised Antibody Heavy andLight Chain Variable Region Genes

[0242] Firstly an overview of the humanisation strategy is set out inthe following text. The purpose of antibody humanisation is to combinethe binding site of a non-human antibody into the supporting frameworkof a human antibody while maintaining the characteristic antigen bindingaffinity and specificity properties of the parent antibody. Thefeasibility of such antibody engineering is a consequence of the closesequence and structural homology of immunoglobulins from differentmammalian species.

[0243] In its most basic form the approach involes the transfer of thesix hypervariable regions or complementarity determing regions (CDRs)from one antibody Fv region to another, as first described in Jones etalNature (1986) 321 522-525. However, experience has shown that inaddition to the CDRs it is often necessary that amino acids in theantibody framework also need to be transferred for the process to besuccessful since such residues sometimes appear to contact and influencethe conformation of the CDR loops.

[0244] In the case of the 806.077 antibody an “Initial” humanisedversion of the antibody was made which comprises the six murine CDRs anda number of framework residue substitutions. This construct was used asa template from which further variants (Examples 12-47) were made byintroducing additional “murine” residue substitutions. The rest of thisExample describes the Initial humanised construct in detail.

[0245] The human antibody heavy chain variable region NEWM (Poljak, R. Jet al (1974) PNAS 71 3440-3444) and the light chain kappa variableregion REI (Palm, W and Hilschmann, N. Z. (1975) Physiol. Chem. 356167-191 were chosen to form the acceptor human antibody framework.Numerous examples of successful humanisations using this Fv frameworkhave been described in the literature and the 3 dimensional structure ofthese two protein domains has been solved. Based on comparison of themurine 806.077 heavy and light chain variable region protein sequenceswith their closest related Kabat murine subgroup concensus sequences(and the individual sequence members) and the human NEWM and REI proteinsequences, individual DNA sequences were designed to encode for theInitial humanised antibody which incorporated the murine CDRs and anyadditional framework substitutions considered to be of importance.

[0246] The murine 806.077 CDR sequences incorporated are described inSEQ ID NOs: 26; 27 and 28 for the light chain variable region and arefound at positions 24-34 (CDR1), 50-56 (CDR2) and 89-97 (CDR3)repectively. The CDRs incorporated in the heavy chain variable regionare described in SEQ ID NOs: 29, 31 and 32 being at positions 31-35(CDR1), 50-65 (CDR2), 95-102 (CDR3) respectively (using Kabatnomenclature). In the heavy chain variable region the additional changesV24A; S27F; T28N; F29I; S30K; .V71A; A92H; R93V (Kabat nomenclature)were made to the NEWM framework and in the light chain variable regionno additional framework changes were made.

[0247] Individual synthetic DNA sequences were designed to encode forthe initial version of the 806.077 humanised antibody heavy(806.077HuVH1) and light chain (806.077HuVK1) variable regions in whichthe CDRs and any additional framework residue changes were incorporated.The antibody variable gene sequences shown in SEQ ID NOS: 48 and 53 maybe prepared by a variety of methods including those described by Edwards(1987) Am. Biotech. Lab. 5, 38-44, Jayaraman et al. (1991) Proc. Natl.Acad. Sci. USA 88, 4084-4088, Foguet and Lubbert (1992) Biotechniques13, 674-675 and Pierce (1994) Biotechniques 16, 708. Preferably, the DNAsequences shown in SEQ ID NOS: 48 and 53 are prepared by a PCR methodsimilar to that described by Jayaraman et al. (1991) Proc. Natl. Acad.Sci. USA 88, 4084-4088.

[0248] The humanised 806.077 antibody variable light chain gene sequence(SEQ ID NO: 49 and 50) was inserted, in frame, by cloning into thepNG3-Vkss-HuCK-Neo (NCIMB no. 40799) expression vector. To achieve this,the synthetic PCR DNA fragment encoding the humanised variable lightchain gene (SEQ ID NO: 48) was digested with SacII and XhoI restrictionenzymes and cloned into the similarly restricted pNG3-Vkss-HuCK-Neovector which contained the VK signal sequence and HuCK constant regioncoding sequences. The DNA and protein sequence of completed humanised806.077 light chain sequence (806.077HuVK1-HuCK) produced, together withits signal sequence, are shown in SEQ ID NOS: 51 and 52 respectively andthe vector named pNG3-Vkss-806.077HuVK1-HuCK-Neo.

[0249] Similarly, for the humanised antibody heavy chain, the humanisedvariable heavy chain gene sequence SEQ ID NO: 54 and 55 was inserted, inframe, by cloning directly into the pNG4-VHss-HuIgG2CH1′ (NCIMB no.40797) expression vector. To achieve this, the synthetic PCR fragmentobtained for the humanised heavy chain gene (SEQ ID NOS: 53) wasdigested with EcoRI and SacI restriction enzymes and cloned into thesimilarly restricted pNG4-VHss-HuIgG2CH1′ vector which contained the VHsignal sequence and HuIgG2 CH1′ constant region coding sequences. TheDNA and protein sequence of completed humanised 806.077 Fd heavy chainsequence (806.077HuVH1-HuIgG2 Fd) produced, together with its signalsequence, are shown in SEQ ID NOS: 56 and 57 respectively and the vectornamed pNG4-VHss-806.077HuVH1-HuIgG2 CH1′.

[0250] The Initial humanised antibody construct was produced byconstructing a co-expression plasmid containing both 806.077 HuVK1 lightchain and 806.077 HuVH1 heavy chain variable region antibody genes. Theplasmid pNG3-Vkss-806.077HuVK1-HuCK-Neo vector, which contains thehumanised light chain variable region HuVK1 (SEQ ID NOS: 49 and 50) wasdigested using the restriction enzymes BamHI and SalI and the vector runon a 1% agarose gel, the vector band was excised and purified. Theplasmid pNG4-VHss-806.077HuVH1-HuIgG2 CH1′ (which contains the humanised806.077HuVH1 heavy chain variable region (SEQ ID NOS: 56 and 57)) wasdigested using the restriction enzymes BglII and SalI, the reaction runon a 2% agarose and the fragment band excised and purified. The DNAfragment recovered was subsequently ligated into the preparedpNG3-Vkss-806.077HuVK1-HuCK-Neo vector to produce clones of the desiredHuVH1/HuVK1 co-expression vector.

[0251] These constructs were transfected into NS0 myeloma cells (ECACCNo. 85110503) via standard techniques of electroporation andtransfectants selected for the property of G418 antibiotic resistance.The clones obtained were tested for both antibody expression in theanti-human antibody Fd ELISA and CEA binding ELISA assays describedbelow.

[0252] For the CEA ELISA each well of a 96 well immunoplate (NUNCMAXISORB™) was coated with 50 ng CEA in 50 mM carbonate/bicarbonatecoating buffer pH9.6 (buffer capsules—Sigma C3041) and incubated at 4°C. overnight. The plate was washed three times with PBS+0.05% Tween 20and then blocked 150 μl per well of 1% BSA in PBS +0.05% Tween 20 for 1hour at room temperature. The plate was washed as previously described,100 μl of test sample added per well and incubated at room temperaturefor 2 hours. Again the plate was washed three times with PBS+0.05% Tween20, 100 μl per well of a 1/500 dilution of HRPO-labelled goat anti-humankappa antibody (Sigma A 7164) was added, in 1% BSA in PBS-Tween 20 andincubated at room temperature on a rocking platform for at least 1 hour.The plate was washed as before and then once more with PBS. To detectbinding add 100 μl per well developing solution (one capsule ofphosphate-citrate buffer—Sigma P4922—dissolved in 100 mls H₂O to whichis added one 30 mg tablet o-phenylenediamine dihydrochloride—SigmaP8412) and incubated for up to 15 minutes. The reaction was stopped byadding 75 μl 2M H₂SO₄, and absorbance read at 490 nm.

[0253] In the anti-human antibody Fd ELISA, each well of a 96 wellimmunoplate was coated with 1.2 μg sheep anti-human Fd antibody (BindingSite PC075) in 50 mM carbonate/bicarbonate coating buffer pH9.6 (buffercapsules—Sigma C3041) and incubated at 4° C. overnight. The plate waswashed three times with PBS+0.05% Tween 20 and then blocked with 150 μlper well of 1% BSA in PBS+0.05% Tween 20 for 1 hour at room temperature.The plate was washed as previously described, 100 μl of test sampleadded per well and incubated at room temperature for 2 hours. Again theplate was washed three times with PBS+0.05% Tween 20, 100 μl per well ofHRPO-labelled goat anti-human kappa antibody (Sigma A 7164) was added in1% BSA in PBS-Tween 20 and incubated at room temperature on a rockingplatform for at least 1 hour. Wash plate as before and then once morewith PBS. To detect binding, developing solution etc was added asdescribed above for the CEA binding assay.

[0254] The clones found to show the best expression and CEA bindinglevels were selected for further expansion into 24 well plates andre-tested. The best clone according to these assay criteria was selectedand expanded such that a one liter production was undertaken , which wasseeded using a 1:10 dilution of a confluently grown culture (i.e. 100mls into 900 mls of fresh culture medium) and the grown for a further 14days. The human F(ab′)₂ antibody fragment was then purified from theculture supernatant as described in Example 102.

EXAMPLES 12-38 Further Combinations of Humanised Heavy and Light ChainVariable Region Gene Variants: Construction of 806.077 Humanised Heavyand Light Variable Region Variants

[0255] The Initial humanised 806.077 variable region genes were alsoused for the subsequent construction of further gene constructs whichcontained additional murine framework residues. Modifications of thegene sequences were achieved (in the majority of cases) by cassettemutagenesis. In this technique part of the original gene was removed viarestriction with two appropriate unique enzymes from the completeplasmid vector and then replaced by a double stranded DNA cassette(consisting of two complementary oligonucleotides hybridised together toform a DNA fragment with the appropriate cohesive ends) by directligation into the prepared plasmid thus reconstituting the gene but nowcontaining desired DNA changes. Further combinations of mutations withineither the heavy or light chain could be also be produced by simple DNAfragment exchanges between the appropriate variants by utilising theavailable unique restriction enzyme sites.

[0256] Three further variants of the humanised light chain variableregion were produced in addition to the original sequence HuVK1 (SEQ IDNO: 49 and 50) and these were called HuVK2, HuVK3 and HuVK4 repectively.The light chain variable region variant HuVK2 was a modification of theoriginal HuVK1 coding sequence in order to produce the amino acid changeM4L (Kabat nomenclature), with the gene (SEQ ID NO: 49) being mutated bycassette mutagenesis. The plasmid pNG3-Vkss-806.077HuVK1-HuCK-Neo (whichcontains the complete humanised light chain (SEQ ID NOS: 49 and 50) wasdigested using the restriction enzymes SacII and NheI. The digest wasthen loaded on a 2% agarose gel and the excised fragment separated fromthe remaining vector. The vector DNA was then excised from the gel,recovered and stored at −20° C. until required. Two oligonucleotides(containing the desired base changes) were designed and synthesised (SEQID NO: 58 and 59). These two oligonucleotides were hybridised by adding200 pmoles of each oligonucleotide into a total of 30 μl of H₂O, heatingto 95° C. and allowing the solution to cool slowly to 30° C. 100 pmolesof the annealed DNA product was then ligated directly into thepreviously prepared vector. This DNA “cassette” exchange produced thedesired HuVK2 DNA and protein sequence (SEQ ID NO: 60 and 61) already inplace in the expression vector pNG3-Vkss-806.077HuVK2-HuCK-Neo.

[0257] Similarly, HuVK3 with the amino acid changes D1Q; Q3V; M4L (Kabatnomenclature) was constructed using synthetic oligonucleotides (SEQ IDNO: 62 and 63) to produce the desired HuVK3 DNA and protein sequence(SEQ ID NO: 64 and 65) again already in place in the expression vectorpNG3-Vkss-806.077HuVK3-HuCK-Neo.

[0258] The light chain variable region variant HuVK4 was produced by adifferent technique, as there were not unique restriction enzyme sitesavailable close to the mutation site. HuVK4, with the amino acid changeL47W, was produced by a PCR mutagenesis technique. The vectorpNG3-806.077HuVK1-HuVK-Neo was used as the template for two PCRreactions (94° C., 90 sec; 55° C., 60 sec; 72° C., 120 sec for 15cycles, all buffers, etc., as previously described). Reaction A used thesynthetic oligonucleotide sequence primers SEQ ID NOS: 66 and 67 andreaction B the synthetic oligonucleotide sequence primers SEQ ID NOS: 68and 69. The products of these PCR reactions (A and B) were fragments oflength 535 base pairs and 205 base pairs respectively. These reactionproducts were run on a 2% agarose gel and separated from any backgroundproducts. Bands of the expected size were excised from the gel andrecovered. Mixtures of varying amounts of the products A and B were madeand PCR reactions performed using the synthetic oligonucleotides SEQ IDNOS: 66 and 68. The resulting product (ca.700 base pairs) was digestedwith the restriction enzymes SacII and XhoI and the cleavage productsseparated on a 2% agarose gel. The band of the expected 310 base pairssize was excised from the gel and recovered. This fragment was thenligated into the vector pNG3-806.077HuVK1-HuVK-Neo vector (which hadbeen previously cut with the restriction enzymes SacII/XhoI andsubsequently isolated) and thus created the desired HuVK4 DNA andprotein sequence (SEQ ID NO: 70 and 71) within the expression vectorpNG3-Vkss-806.077HuVK4-HuCK-Neo.

[0259] Six further variants of the humanised heavy chain variable regionwere produced in addition to the original HuVH1 sequence (SEQ ID NO: 54and 55) and these were called HuVH2 to HuVK7 respectively. The heavychain variable region variant HuVH2 was a modification of the originalHuVH1 coding sequence in order to produce the amino acid change G49A(Kabat nomenclature), with the gene (SEQ ID NO: 54) being mutated bycassette mutagenesis. The plasmid pNG4-VHss-806.077HuVH1-HuIgG2 CH1′(which contains the complete humanised, IgG2 heavy chain Fd (SEQ ID NOS:56 and 57) was digested using the restriction enzymes StuI and NotI. Thedigest was then loaded on a 2% agarose gel and the excised fragmentseparated from the remaining vector. The vector DNA was then excisedfrom the gel, recovered and stored at −20° C. until required. Twooligonucleotides were designed, synthesised (SEQ ID NO: 72 and 73),hybridised and the product ligated directly into the previously preparedvector. This DNA “cassette” exchange produced the desired HuVH2 DNA andprotein sequence (SEQ ID NO: 74 and 75) already in place in theexpression vector pNG4-VHss-806.077HuVH2-HuIgG2 CH1′.

[0260] Similarly, HuVH3 with the amino acid changes T73S; F78A (Kabatnomenclature) was constructed using synthetic oligonucleotides (SEQ IDNO: 76 and 77), however, in this case, the vectorpNG4-VHss-806.077HuVH1-HuIgG2 CH1′ was digested using the restrictionenzymes NotI and SacII. The synthetic DNA cassette was ligated directlyinto the previously prepared vector to produce the desired HuVH3 DNA andprotein sequence (SEQ ID NO: 78 and 79) in the expression vectorpNG4-VHss-806.077HuVH3-HuIgG2 CH1′.

[0261] HuVH4 with the amino acid changes G49A; T73S; and F78A (Kabatnomenclature) combines the HuVH2 (SEQ ID NO: 74 and 75) and HuVH3 (SEQID NO: 78 and 79) variants. This was achieved by digesting thepNG4-VHss-806.077HuVH3-HuIgG2 CH1′ vector with the enzymes NotI and NheIand isolating the ca. 200 base pairs NotI/NheI restriction fragmentafter separation on a 2% agarose gel. The fragment was recovered andsubsequently ligated into the pNG4-VHss-806.077HuVH2-HuIgG2 CH1′ vector(which had been digested with the same Not I and NheI restrictionenzymes and the vector fragment purified). The resulting clonescontained the desired HuVH4 DNA and protein sequence ((SEQ ID NO: 80 and81) in the expression vector pNG4-VHss-806.077HuVH4-HuIgG2 CH1′.

[0262] HuVH5 with the amino acid changes V67A (Kabat nomenclature) wasconstructed using synthetic oligonucleotides (SEQ ID NO: 82 and 83).Again, the vector pNG4-VHss-806.077HuVH1-HuIgG2 CH1′ was digested usingthe restriction enzymes NotI and SacII. The synthetic DNA cassette wasligated directly into the previously prepared vector to produce thedesired HuVH5 DNA and protein sequence (SEQ ID NO: 84 and 85) in theexpression vector pNG4-VHss-806.077HuVH5-HuIgG2 CH1′.

[0263] HuVH6 with the amino acid changes V67A; T73S and F78A (Kabatnomenclature) was constructed using synthetic oligonucleotides (SEQ IDNO: 86 and 87) and for this mutant the vectorpNG4-VHss-806.077HuVH1-HuIgG2 CH1′ was digested using the restrictionenzymes NotI and SacII. The synthetic DNA cassette was ligated directlyinto the previously prepared vector to produce the desired HuVH6 DNA andprotein sequence (SEQ ID NO: 88 and 89) in the expression vectorpNG4-VHss-806.077HuVH6-HuIgG2 CH1′.

[0264] HuVH7 with the amino acid changes G49A; V69A; T73S; and F78A(Kabat nomenclature) combines the HuVH2 (SEQ ID NO: 74 and 75) and HuVH6(SEQ ID NO: 88 and 89) variants. This was achieved by digesting thepNG4-VHss-806.077HuVH6-HuIgG2 CH1′ vector with the enzymes NotI and NheIand isolating the ca. 200 base pairs NotI/NheI restriction fragmentafter separation on a 2% agarose gel. The fragment was recovered andligated into the pNG4-VHss-806.077HuVH2-HuIgG2 CH1′ vector (which hadbeen digested with the same Not I and NheI restriction enzymes and thevector fragment purified). The resulting clones contained the desiredHuVH7 DNA and protein sequence (SEQ ID NO: 90 and 91) in the expressionvector pNG4-VHss-806.077HuVH7-HuIgG2 CH1′.

[0265] Combinations of such humanised heavy and light chain variablegene variants were made by excising the heavy chain Fd gene variantexpression cassette (including both promoter and gene excised as aBglII/SalI fragment) and cloning this fragment into the BamHI/SalI sitesof the light chain variant vector to produce a co-expression vectorconstruct. A listing of the possible combinantions of variants based onthe humanised heavy and light chain variants previously described isshown in the table below. TABLE Combinations of humanised heavy andlight chain variable region variants. Heavy chain Light chain Examplevariable SEQ ID variable SEQ ID No. region NOS: region NOS:Co-expression Plasmid Vector 11 HuVH1 54 and 55 HuVK1 49 and 50 pNG806HuVH1/HuVK1/HulgG2 12 HuVH1 54 and 55 HuVK2 60 and 61 pNG806HuVH1/HuVK2/HulgG2 13 HuVH1 54 and 55 HuVK3 64 and 65 pNG806HuVH1/HuVK3/HulgG2 14 HuVH1 54 and 55 HuVK4 70 and 71 pNG806HuVH1/HuVK4/HulgG2 15 HuVH2 74 and 75 HuVK1 49 and 50 pNG806HuVH2/HuVK1/HulgG2 16 HuVH2 74 and 75 HuVK2 60 and 61 pNG806HuVH2/HuVK2/HulgG2 17 HuVH2 74 and 75 HuVK3 64 and 65 pNG806HuVH2/HuVK3/HulgG2 18 HuVH2 74 and 75 HuVK4 70 and 71 pNG806HuVH2/HuVK4/HulgG2 19 HuVH3 78 and 79 HuVK1 49 and 50 pNG806HuVK3/HuVK1/HulgG2 20 HuVH3 78 and 79 HuVK2 60 and 61 pNG806HuVH3/HuVK2/HulgG2 21 HuVH3 78 and 79 HuVK3 64 and 65 pNG806HuVH3/HuVK3/HulgG2 22 HuVH3 78 and 79 HuVK4 70 and 71 pNG806HuVH3/HuVK4/HulgG2 23 HuVH4 80 and 81 HuVK1 49 and 50 pNG806HuVH4/HuVK1/HulgG2 24 HuVH4 80 and 81 HuVK2 60 and 61 pNG806HuVH4/HuVK2/HulgG2 25 HuVH4 80 and 81 HuVK3 64 and 65 pNG806HuVH4/HuVK3/HulgG2 26 HuVH4 80 and 81 HuVK4 70 and 71 pNG806HuVH4/HuVK4/HulgG2 27 HuVH5 84 and 85 HuVK1 49 and 50 pNG806HuVH5/HuVK1/HulgG2 28 HuVH5 84 and 85 HuVK2 60 and 61 pNG806HuVH5/HuVK2/HulgG2 29 HuVH5 84 and 85 HuVK3 64 and 65 pNG806HuVH5/HuVK3/HulgG2 30 HuVH5 84 and 85 HuVK4 70 and 71 pNG806HuVH5/HuVK4/HulgG2 31 HuVH6 88 and 89 HuVK1 49 and 50 pNG806HuVH6/HuVK1/HulgG2 32 HuVH6 88 and 89 HuVK2 60 and 61 pNG806HuVH6/HuVK2/HulgG2 33 HuVH6 88 and 89 HuVK3 64 and 65 pNG806HuVH6/HuVK3/HulgG2 34 HuVH6 88 and 89 HuVK4 70 and 71 pNG806HuVH6/HuVK4/HulgG2 35 HuVH7 90 and 91 HuVK1 49 and 50 pNG806HuVH7/HuVK1/HulgG2 36 HuVH7 90 and 91 HuVK2 60 and 61 pNG806HuVH7/HuVK2/HulgG2 37 HuVH7 90 and 91 HuVK3 64 and 65 pNG806HuVH7/HuVK3/HulgG2 38 HuVH7 90 and 91 HuVK4 70 and 71 pNG806HuVH7/HuVK4/HulgG2

[0266] Analogously with Example 11, Example 14 was produced byconstructing a co-expression plasmid containing both the 806.077 HuVK4light chain and the 806.077 HuVH1 heavy chain variable region antibodygenes. In this case, the plasmid the pNG3-Vkss-806.077HuVK4-HuCK-Neovector, which contains the humanised light chain variable region HuVK1(SEQ ID NOS: 70 and 71) was digested using the restriction enzymes BamHIand SalI and the vector run on an 1% agarose gel and the vector bandpurified. The plasmid pNG4-VHss-806.077HuVH1-HuIgG2 CH1′ (which containsthe humanised 806:077 HuVH1 heavy chain variable region (SEQ ID NOS: 56and 57) was digested using the restriction enzymes BglII and SalI, thereaction run on an 2% agarose and the fragment band excised andpurified. The DNA fragment recovered was ligated into the preparedpNG3-Vkss-806.077HuVK4-HuCK-Neo vector to produce clones of the desiredHuVH1/ HuVK4 co-expression vector.

[0267] As described in Example 11, these constructs were transfectedinto NS0 myeloma cells (ECACC No. 85110503) via standard techniques ofelectroporation and transfectants selected for the property of G418resistance. The clones obtained were tested for both antibody expressionin anti-human antibody Fd ELISA and CEA binding ELISA assays. Clonesfound to show the best expression and CEA binding levels were selected,expanded and product expressed. Human F(ab′)₂ antibody fragment was thenpurified from the culture supernatant as described in Example 102.

EXAMPLE 39-47 Expression of Humanised F(ab′)₂ Fragments with VariousClasses of Human Heavy Chain Constant Regions

[0268] Other classes of chimaeric heavy chain Fd constructs may be used.Accordingly, additional variants of the heavy chain vectors have beenmade which contain either HuIgG1CH1′ (SEQ ID NOS: 20 and 21) orHuIgG3CH1′ (SEQ ID NOS: 24 and 115), the constant regions of which aresubstituted for the HuIgG2CH1′ gene (SEQ ID NOS: 22 and 23). The vectorscreated were pNG4-VHss-HuIgG1 CH1′ pNG4-VHss-HuIgG3 CH1′ respectively.The heavy chain antibody variable region in question can be excised fromthe appropriate pNG4-VHss-“VH variable region”-HuIgG2 CH1′ plasmid bydigestion with EcoRI and SacI restriction enzymes and cloned into thesimilarly restricted pNG4-VHss-HuIgG1CH1′ or pNG4-VHss-HuIgG3 CH1′vector and thus produce a completed heavy chain Fd sequence. Asdescribed above, once the individual heavy and light chain sequences areconstructed, a heavy chain Fd gene expression cassette (including bothpromoter and gene can be excised by restriction digestion and thefragment cloned between into the appropriate sites of the light chainvector to produce the final co-expression vector. The table belowdescribes Examples 39-47 in which various heavy and light chain variableregions have been combined with a number of different classes of humanheavy chain constant regions.

[0269] In Example 44, the vector pNG4-VHss-HuIgG3 CH1′ was digested withthe restriction enzymes EcoRI and SacI restriction enzymes and thevector fragment isolated as previously described. The HuVH1 heavy chainantibody variable region (SEQ ID NOS: 54 and 55) was excised from thepNG4-VHss-806.077HuVH1-HuIgG2 CH1′ plasmid by digestion with EcoRI andSacI restriction enzymes and the fragment cloned into the similarlyrestricted pNG4-VHss-HuIgG3 CH1′ vector to produce a completed humanisedIgG3 heavy chain Fd sequence (SEQ ID NOS: 94 and 95) in the completedvector pNG4-VHss-806.077HuVH1-HuIgG3 CH1′. The heavy chain Fd geneexpression cassette (including both promoter and gene) was excised as aBglII/SalI fragment and cloned into the BamHI/SalI sites of the lightchain vector pNG3-Vkss-806.077HuVK1-HuCK-Neo (containing the HuVK1-HuCKhumanised light chain SEQ ID NOS: 51 and 52) which had been digestedusing the restriction enzymes BamHI and SalI, run on an 1% agarose, thevector band purified. This produced a co-expression vector (pNG806HuVH1/HuVK3/HuIgG3) from which the humanised806.077HuVH1/HuVK1-HuIgG3/Kappa.Fd antibody fragment could be expressed.TABLE SEQ Example Humanised ID Humanised SEQ ID No. heavy chain NOSlight chain NOS Co-expression Plasmid Vector 39 HuVH1-HulgG1 92 andHuVK1-HuCK 51 and 52 Png 806HuVH1/HuVK1/HulgG1 93 40 HuVH1-HulgG2 56 andHuVK1-HuCK 51 and 52 pNG 806HuVH1/HuVK1/HulgG2 57 41 HuVH1-HulgG3 94 andHuVK1-HuCK 51 and 52 pNG 806HuVH1/HuVK1/HulgG3 95 42 HuVH1-HulgG1 92 andHuVK3-HuCK 96 and 97 pNG 806HuVH1/HuVK3/HulgG1 93 43 HuVH1-HulgG2 56 andHuVK3-HuCK 96 and 97 pNG 806HuVH1/HuVK3/HulgG2 57 44 HuVH1-HulgG3 94 andHuVK3-HuCK 96 and 97 pNG 806HuVH1/HuVK3/HulgG3 95 45 HuVH1-HulgG1 92 andHuVK4-HuCK 98 and 99 pNG 806HuVH1/HuVK4/HulgG1 93 46 HuVH1-HulgG2 56 andHuVK4-HuCK 98 and 99 pNG 806HuVH1/HuVK4/HulgG2 57 47 HuVH1-HulgG3 94 andHuVK4-HuCK 98 and 99 pNG 806HuVH1/HuVK4/HulgG3 95

[0270] In Example 47, the vector pNG4-VHss-HuIgG3 CH1′ was digested withEcoRI and SalI restriction enzymes and the vector fragment isolated. TheHuVH1 heavy chain antibody variable region (SEQ ID NOS: 54 and 55) wasexcised from the pNG4-VHss-HuVH1-HuIgG2 CH1′ plasmid by digestion withEcoRI and SacI restriction enzymes and the fragment cloned into thesimilarly restricted pNG4-VHss-HuIgG3 CH1′ vector. This produces acompleted humanised IgG3 heavy chain Fd sequence (SEQ ID NOS: 94 and 95)in the completed vector pNG4-VHss-806.077HuVH1-HuIgG3 CH1′. The heavychain Fd gene expression cassette (including both promoter and gene) wasexcised as a BglII/SalI fragment and cloned into the BamHI/SalI sites ofthe light chain vector pNG3-Vkss-806.077HuVK4-HuCK-Neo vector,(containing HuVK4-HuCK humanised light chain SEQ ID NOS: 98 and 99 )which had been digested using the restriction enzymes BamHI and SalI,run on an 1% agarose, the vector band purified. This produced aco-expression vector construct pNG 806HuVH1/HuVK4/HuIgG3 from which thehumanised HuVH1/HuVK1-HuIgG3/Kappa.Fd antibody fragment could beexpressed.

[0271] The other Examples shown in the table above were all produced ina similar manner to that described in the Examples 44 and 47. However,in the case of the constructs containing human IgG1, the finalco-expression vector construction was made by cloning the heavy chain Fdgene expression cassette (including both promoter and gene) excised as aBglII/BamHI fragment (because there is an internal SalI restriction sitein the HuIgG1 CH1′ constant region gene) and cloned into the BamHI siteof the appropriately prepared light chain vector. In this case theorientation of the heavy chain cassette must be checked. This wasachieved by restriction digestion (e.g. with the restriction enzyme HindIII) and agarose gel electrophoresis analysis in which the resultingfragment sizes were viewed relative to comparable fragments from asimilarly digested HuIgG2 version (Examples 11-38). When thefragmentation patterns matched for both constructs we could be sure thatthe heavy chain cassette was in the correct orientation.

[0272] As previously described in Example 11, these constructs weretransfected into NS0 myeloma cells (ECACC No. 85110503) via standardtechniques of electroporation and transfectants selected for theproperty of G418 resistance. The clones obtained were tested for bothantibody expression in the anti-human antibody Fd ELISA and CEA bindingELISA assays and the clones found to show the best expression and CEAbinding levels were selected, expanded and grown for gene expression. Asbefore, the human F(ab′)₂ antibody fragment was then purified from theculture supernatant as described in Example 102.

EXAMPLE 48 Preparation of Humanised 806.077F(ab′)₂-[A248S,G251T,D253K]HCPB Fusion Protein

[0273] This Example describes the preparation of a gene encoding ahumanised Fd heavy chain fragment of 806.077 linked to[A248S,G251T,D253K]HCPB and its co-expression with a gene encoding ahumanised light chain of 806.077 and a gene encoding the pro domain ofhuman carboxypeptidase B to give the F(ab′)₂ protein with a molecule of[A248S,G251T,D253K]HCPB at the C-terminus of each of the heavy chainfragments. The constant and hinge regions of of the humanised Fd heavychain fragment are derived from the human IgG3 antibody isotype. Theexpressed protein is also referred to as antibody-enzyme fusion protein.

[0274] (a) Preparation of a Gene Encoding Humanised Fd Heavy ChainFragment of 806.077 Linked to [A248S, G251T, D253K]HCPB and its Cloninginto pEE6

[0275] A gene encoding humanised 806.077 Fd linked to[A248S,G251T,D253K]HCPB was generated by PCR from pZEN1921 (ReferenceExample 2). A first PCR was set up with template pZEN1921 (2 ng) andoligonucleotides SEQ ID NO: 100 and SEQ ID NO: 101 (100 pM of each) inbuffer (100 μl) containing 10 mM Tris-HCl (pH8.3), 50 mM KCL, 1.5 mMMgCl₂, 0.125 mM each of dATP, dCTP, dGTP and dTTP. The reaction wasincubated at 94° C. for 5 min then thermostable DNA polymerase (2.5 u,0.5 μl) was added and the mixture overlaid with mineral oil (100 μl) andthe reaction mixture incubated at 94° C. for 1 min, 53° C. for 1 min and72° C. for 2.5 min for 25 cycles, plus 10 min at 72° C. The PCR productof 536 base pairs was isolated by electrophoresis on a 1% agarose(Agarose type I, Sigma A-6013) gel followed by excision of the band fromthe gel and isolation of the DNA fragment.

[0276] A second PCR was set up with template IgG3-pBSIIKS+ (8.7 ng,described in Reference Example 4) and oligonucleotides SEQ ID NO: 102and SEQ ID NO: 103 and the 954 base pairs fragment isolated as describedabove. The products from the 2 PCRs were combined (either at 0.2, 1.0 or5.0 ng/μl) in PCR buffer as described above. The mixture was incubatedfor at 94° C. for 5 min then 10 cycles at 94° C. for 1 min and 63° C.for 4 min. Oligos SEQ ID NOS: 101 and 102 (100 pM of each) in PCR buffer(50 μl ) were added. After incubation at 94° C. for 3 min, the mixturewas further incubated at 94° C. for 1.5 min, 53° C. for 2 min and 72° C.for 2 min for 25 cycles plus 10 min at 72° C. In this process, the Gbase at position 508 in SEQ ID NO: 115 was changed to an A base.

[0277] The PCR product of 1434 base pairs was isolated byelectrophoresis on a 1% agarose gel, purified and digested with NheI (20u) and XbaI (80 u) (New England Biolabs Inc.,) in a total volume of 100μl containing 10 mM Tris HCl (pH7.9), 50 mM NaCl, 10 mM MgCl₂, 1 mM DTTand BSA (100 μg/ml) for 4 h at 37° C. The resulting fragment was againisolated by electrophoresis on a 1% agarose gel and purified. In asimilar digestion, vector pNG4-VHss-806.077huVH1-HuIgG2CH1′ (10 μg;Example 11) was cut with NheI and XbaI then calf intestinal alkalinephosphatase (1 μl; New England Biolabs, 10 u/μl) was added to thedigested plasmid to remove 5′ phosphate groups and incubation continuedat 37° C. for a further 30 minutes. Phosphatase activity was destroyedby incubation at 70° C. for 10 minutes. The NheI-XbaI cut plasmid waspurified from an agarose gel. The NheI-XbaI digested PCR product fromabove (about 500 ng) was ligated with the above cut plasmid DNA (about200 ng) in 20 μl of a solution containing 50 mM Tris-Hcl (pH7.8), 10 mMMgCl₂, 10 mM DTT, 1 mM ATP, 50 μg/ml BSA and 400 u T4 DNA ligase (NewEngland Biolabs, Inc) at 25° C. for 4 h. A 1 μl aliquot of the reactionwas used to transform 20 μl of competent E. coli DH5α cells. Transformedcells were plated onto L-agar plus 100 μg/ml ampicillin. Potentialclones containing the gene for humanised 806.077Fd-[A248S,G251T,D253K]HCPB were identified by PCR. Each clone wassubjected to PCR as described above with oligonucleotides SEQ ID NOS:104 and 105. A sample (10 μl) of the PCR reaction was analysed byelectrophoresis on a 1% agarose gel. Clones containing the required genewere identified by the presence of a 512 base pairs PCR product. Clonesproducing the 512 base pairs band were used for DNA minipreps. The DNAsamples were checked by digestion with HindIII and XbaI for the presenceof 3751 base pairs and 1862 base pairs fragments. Clones containingthese fragments on digestion of the DNA with HindIII and XbaI were usedfor large scale plasmid DNA preparation and the sequence of the insertconfirmed by DNA sequence analysis. The sequence of the expected insertis shown in SEQ ID NO: 112 Of the clones examined above, 2 contained theexpected sequence but with a single base mutation. Clone 54 (alsodesignated pMF195) had an T base at position 605 in SEQ ID NO: 112 inplace of the A base, whereas clone 68 (also designated pMF198) had a Cbase at position 1825 instead of the expected T base. The sequence shownin SEQ ID NO: 112 was prepared from pMF195 and and pMF198 by digestingboth (10 μg of each) with XmaI (10 u) and XbaI (100 u) (New EnglandBiolabs) in buffer (100 μl) containing 20 mM Tris acetate (pH7.9) 50 mMpotassium acetate. 10 mM Mg acetate. 1 mM DTT and BSA (100 μg/ml). The215 base pairs fragment from pMF195 and the vector fragment from pMF198(following treatment with alkaline phosphatase) were isolated from a 1%agarose gel and ligated together as described previously. The ligationmix was used to transform competent DH5α cells. The transformed cellswere plated onto L agar plus ampicillin and resulting colonies screenedby digestion of the DNA with XmaI and XbaI for the presence of 5400 basepairs and 215 base pairs fragments. Positive clones were used for largescale plasmid DNA preparation and the sequence of the insert confirmedby DNA sequence analysis. The plasmid containing the 806.077Fd-[A248S,G251T,D253K]HCPB gene from clone number 102 was named pMF213.The HindIII-XbaI fragment from pMF213 was cloned into pEE6 [this is aderivative of pEE6.hCMV—Stephens and Cockett (1989) Nucleic AcidsResearch 17, 7110—in which a HindIII site upstream of the hCMV promoterhas been converted to a BglII site] in DH5α (screened by PCR witholigonucleotides SEQ ID NOS: 106 and 107 for a 2228 base pairs insert)to give pMF221.

[0278] (b) Preparation of a Co-Expression Vector for Expression ofAntibody-Enzyme Fusion Protein

[0279] To generate vectors capable of expressing the antibody-enzymefusion protein in eukaryotic cells, the GS-System™ (Celltech Biologics)was used (WO 87/04462, WO 89/01036, WO 86/05807 and WO 89/10404). Theprocedure requires cloning the humanised antibody light chain gene intothe HindIII-XmaI region of vector pEE14. This vector is described byBebbington in METHODS: A Companion to methods in Enzymology (1991) 2,136-145. To construct the expression vector, plasmids pEE14 andpNG3-VKss-806.077HuVK4-HuCK-Neo (Example 14) were digested with HinIIIXmaI as described above. The appropriate vector (from pEE14) and insert(732 base pairs from pNG3-VKss-806.077HuVK4-HuCK-Neo) from each digestwere isolated from a 1% agarose gel and ligated together and used totransform competent DH5α cells. The transformed cells were were platedonto L agar plus ampicillin (100 μg/ml). Colonies were screened byrestriction analysis of isloated DNA for the presence of a 732 basepairs fragment on digestion of the DNA with HindIII and XmaI. Clonesproducing a 732 base pairs restriction fragment were used for largescale plasmid DNA preparation and the sequence of the insert confirmedby DNA sequence analysis. The plasmid containing the humanised lightchain sequence of SEQ ID NO: 70 in pEE14 was namedpEE14-806.077HuVK4-HuCK.

[0280] To make the co-expression vector, pMF221 (10 μg) was cut withBglII (20 u) and SalI (40U) in buffer (100 μl) containing 10 mM Tris-HCl(pH 7.9), 150 mM NaCl, 10 mM MgCl₂, 1 mM DTT and BSA (100 μg/ml) and the4560 base pairs fragment isolated by agarose gel electrophoresis andpurified. Similarly, pEE14-806.077HuVK4-HuCK was cut with BamHI (40 u)and SalI (40 u) and the 9.95 kb vector fragment isolated and ligated tothe BglII-SalI fragment from pMF21 and cloned into DH5α. Colonies werescreened by PCR with 2 sets of oligonucleotides (SEQ ID NOS: 104 and105, and SEQ ID NOS: 108 and 109). Clones giving PCR products of 185base pairs and 525 base pairs respectively were characterised by DNAsequencing. A clone with the correct sequence was named pMF228—lightchain/Fd-mutant HCPB co-expression vector in DH5α. The humanisedFd-mutant HCPB sequence is shown in SEQ ID NO: 1 13. Residues 1 to 19are the signal sequence, residues 20 to 242 are the humanised variableand IgG3 CH1 region, residues 243 to 306 are the IgG3 hinge region andresidues 307 to 613 are the mutant HCPB sequence with the changes atresidues 248, 251 and 253 from the human HCPB sequence. The changes inthe HCPB sequence occur in SEQ ID NO: 113 at postions 554 (Ser), 557(Thr) and 559 (Lys) respectively.

[0281] (c) Preparation of a Vector for Expression of the Pro Domain ofproHCPB

[0282] A second eukaryotic expression plasmid, pEE12 containing a genefor the prepro sequence, for secretion of the pro domain with anadditional C-terminal leucine residue (termed pro-L), of preproHCPB wasprepared as described in Reference Example 17 of International PatentApplication Number WO 96/20011. Plasmid pMF161 was prepared by PCR frompMF18 as described for the unmodified prepro sequence, but usingoligonucleotides SEQ ID NOS: 110 and 111. The 359 base pairs fragmentwas cloned into pBluescript to give pMF141 and subsequently into pEE12to give pMF161. The protein sequence of pro-L is shown in SEQ ID NO:114.

[0283] (d) Expression of Antibody-Enzyme Fusion Protein in EukaryoticCells

[0284] For expression in eukaryotic cells, vectors containing genescapable of expressing the antibody enzyme-fusion protein (pMF228) andthe pro-L sequence (pMF161) were co-transfected into COS-7 cells. COScells are an African green monkey kidney cell line, CV-1, transformedwith an origin-defective SV40 virus and have been widely used forshort-term transient expression of a variety of proteins because oftheir capacity to replicate circular plasmids containing an SV40 originof replication to very high copy number. There are two widely availableCOS cell clones, COS-1 and COS-7. The basic methodology for transfectionof COS cells is described by Bebbington in Methods: A Companion toMethods in Enzymology (1991) 2, p. 141. For expression of HCPB, theplasmid vectors pMF48 and pMF67 (2 μg of each) were used to transfectthe COS-7 cells (2×10⁵) in a six-well culture plate in 2 ml Dulbecco'sModified Eagle's Medium (DMEM) containing 10% heat inactivated foetalcalf serum (FCS) by a method known as lipofection—cationiclipid-mediated delivery of polynucleotides [Felgner et al. in Methods: ACompanion to Methods in Enzymology (1993) 5, 67-75]. The cells wereincubated at 37° C. in a CO₂ incubator for 20 h. The mix of plasmid DNAin serum-free medium (200 μl) was mixed gently with LIPOFECTIN™ reagent(12 μl) and incubated at ambient temperature for 15 min. The cells werewashed with serum-free medium (2 ml). Serum-free medium (600 μl) wasadded to the DNA/LIPOFECTIN™ and the mix overlaid onto the cells whichwere incubated at 37° C. for 6 h in a CO₂ incubator. The DNA containingmedium was replaced with normal DMEM containing 10% FCS and the cellsincubated as before for 72 h. Cell supernatants (diluted 1:10 with0.025M Tris-HCl pH7.5; 125 μl) were analysed for activity againstHipp-Glu (5 h assay, in a total volume of 250 μl) essentially asdescribed in Example 103. The diluted supernatant resulted in 18.4%hydrolysis of the Hipp-Glu substrate.

[0285] Alternatively, the unmodified pro domain (from plasmid pMF67described in Reference Example 17 of International Patent ApplicationNumber WO 96/20011) can be used in place of the pro-L expression plasmidin the above experiment.

[0286] Large scale expression of proteins from COS cells is described byRidder et al. (1995) in GENE 166, 273-276 and by Blasey et al. (1996) inCRYOTECHNOLOGY 18, 183-192.

[0287] For stable expression in CHO cells, the procedures described byBebbington in METHODS: A Companion to Methods in Enzymology (1991) 2,136-145 using GS selection with 25 μM and 50 μM MSX are followed.Alternatively, lipofection, essentially as described above fortransfection of COS cells may also be used to transfect CHO cells. Thecells are transfected with a mixture of plasmids pMF228 and pMF161 orpMF228 and pMF67. Supernatants from surviving colonies are screened byCEA ELISA (described in Example 11) and Western analysis (describedbelow) for the presence of a 170 kDa band corresponding to the requiredantibody enzyme fusion protein. Supernatants, suitably diluted, are alsoscreened for enzyme activity as described in Example 103. Coloniesexpressing the desired antibody enzyme fusion protein are cultured atthe required scale (see for Example the publication by M E Reff(1993) inCurrent Opinion in Biotechnology 4, 573-576 and references citedtherein) and fusion protein purified from cell culture supernatant byone or more of the methods described in Example 102.

[0288] (e) Western Analysis

[0289] Western blot analysis was performed as described as follows.Aliquots (20 μl ) of each supernatant sample were mixed with an equalvolume of sample buffer (62.5 mM Tris, pH6.8, 1% SDS, 10% sucrose and0.05% bromophenol blue) with and without reductant. The samples wereincubated at 65° C. for 10 minutes before electrophoresis on a 8-18%acrylamide gradient gel (EXCEL™ gel system from Pharmacia BiotechnologyProducts) in a MULTIPHOR™ II apparatus (LKB Produkter AB) according tothe manufacturer's instructions. After electrophoresis, the separatedproteins were transfered to a membrane (HYBOND™ C-Super, AmershamInternational) using a NOVABLOT™ apparatus (LKB Produkter AB) accordingto protocols provided by the manufacturer. After blotting, the membranewas air dried.

[0290] The presence of antibody fragments was detected by the use of ananti-human kappa antibody (Sigma A7164, goat anti-human Kappa lightchain peroxidase conjugate) used at 1:2500 dilution. The presence ofhuman antibody fragments was visualised using a chemiluminescence system(ECL™ detection system, Amersham International).

EXAMPLES 49-74 Preparation of Other Humanised 806.077 F(ab′)₂-MutantHCPB Fusion Proteins

[0291] These Examples describe preparation of genes encoding humanisedFd heavy chain fragments of 806.077 linked to a mutant HCPB (D253K;G251T,D253K; A248S,G251T,D253K) and their co-expression with a geneencoding a humanised light chain of 806.077 and a gene encoding the prodomain of human carboxypeptidase B to give the F(ab′)₂ protein with amolecule of mutant HCPB at the C-terminus of each of the heavy chainfragments. The constant and hinge regions of of the humanised Fd heavychain fragment are derived from the human IgG1 or IgG2 or IgG3 antibodyisotype. The expressed proteins are also referred to as antibody-enzymefusion proteins.

[0292] The procedures described in Example 48 are repeated with theappropriate sequences derived from the table shown below.Oligonucleotides for PCR constructions and clone screening are readilyderived from the appropriate sequences.

[0293] To change the mutant HCPB sequence, the PCR template, plasmidpZEN1921, in part (a) of Example 48 is replaced with pZEN1860 for[G251T,D253K]HCPB (described in Reference Example 1) or pIC11713 for[D253K]HCPB (described in International Patent Application Number WO96/20011).

[0294] To change the antibody heavy chain constant and hinge region, thePCR template, vector IgG3-pBSIIKS+, in part (a) of Example 48 isreplaced with pNG4-VHss-HuIgG1CH1′ (described in Examples 39-47) orpNG4-VHss-HuIgG2CH1′ (NCIMB No.40797).

[0295] To change the humanised antibody light chain sequence, the vectorpEE14-806.077HuVK4-HuCK in part (b) of Example 48 is replaced withpEE14-806.077HuVK1-HuCK or pEE14-806.077HuVK3-HuCK. The vectorspEE14-806.077HuVK1-HuCK and pEE14-806.077HuVK3-HuCK are prepared asdescribed for pEE14-806.077HuVK4-HuCK in part (b) of Example 48 butusing the 732 base pairs HindIII-Xmal fragment frompNG-VHss-806.077HuVK1-Neo and pNG-VHss-806.077HuVK3-Neo respectively(described in Examples 12-38) in place of the HindIII-XmaI fragment frompNG-VHss-806.077HuVK4-Neo.

[0296] Antibody-enzyme fusion protein variants for each Example areshown in the table below. TABLE Ex- am- ple Humanised Humanised MutantHCPB No. Heavy chain Light chain Enzyme 49 HuVH1-HulgG3 HuVK4-HuCk[D253K]HCPB 50 HuVH1-HulgG3 HuVK4-HuCK [G251T,D253K]HCPB 51 HuVH1-HulgG3HuVK1-HuCK [A248S,G251T,D253K]HCPB 52 HuVH1-HulgG3 HuVK1-HuCK[D253K]HCPB 53 HuVH1-HulgG3 HuVK1-HuCK [G251T,D253K]HCPB 54 HuVH1-HulgG3HuVK3-HuCK [A248S,G251T,D253K]HCPB 55 HuVH1-HulgG3 HuVK3-HuCK[D253K]HCPB 56 HuVH1-HulgG3 HuVK3-HuCK [G251T,D253K]HCPB 57 HuVH1-HulgG1HuVK4-HuCK [A248S,G251T,D253K]HCPB 58 HuVH1-HulgG1 HuVK4-HuCK[D253K]HCPB 59 HuVH1-HulgG1 HuVK4-HuCK [G251T,D253K]HCPB 60 HuVH1-HulgG1HuVK1-HuCK [A248S,G251T,D253K]HCPB 61 HuVH1-HulgG1 HuVK1-HuCK[D253K]HCPB 62 HuVH1-HulgG1 HuVK1-HuCK [G251T,D253K]HCPB 63 HuVH1-HulgG1HuVK3-HuCK [A248S,G251T,D253K]HCPB 64 HuVH1-HulgG1 HuVK3-HuCK[D253K]HCPB 65 HuVH1-HulgG1 HuVK3-HuCK [G251T,D253K]HCPB 66 HuVH1-HulgG2HuVK4-HuCK [A248S,G251T,D253K]HCPB 67 HuVH1-HulgG2 HuVK4-HuCK[D253K]HCPB 68 HuVH1-HulgG2 HuVK4-HuCK [G251T,D253K]HCPB 69 HuVH1-HulgG2HuVK1-HuCK [A248S,G251T,D253K]HCPB 70 HuVH1-HulgG2 HuVK1-HuCK[D253K]HCPB 71 HuVH1-HulgG2 HuVK1-HuCK [G251T,D253K]HCPB 72 HuVH1-HulgG2HuVK3-HuCK [A248S,G251T,D253K]HCPB 73 HuVH1-HulgG2 HuVK3-HuCK[D253K]HCPB 74 HuVH1-HulgG2 HuVK3-HuCK [G251T,D253K]HCPB

EXAMPLE 75 Preparation of [A248S,G251T,D253K]HCPB-(Humanised806.077)F(ab′)₂ Fusion Protein

[0297] This example describes the preparation of a gene encodingpro[A248S,G251T,D253K]HCPB linked to a humanised (version 1 VH withHuman IgG3) Fd heavy fragment of antibody 806.077, and its co-expressionwith a gene encoding a humanised light chain (version 4 VK with CK) ofthe 806.077 antibody. This gives the F(ab′)₂ protein with a molecule ofthe pro-[A248S,G251T,D253K]HCPB at the N-terminus of each of the heavychain fragments. The enzyme is activated by the enzymatic removal of thepro domain using trypsin.

[0298] Standard molecular biology techniques, such as restriction enzymedigestion, ligation, kinase reactions, dephosphorylation, polymerasechain reaction (PCR), bacterial transformations, gel electrophoresis,buffer preparation and DNA generation, purification and isolation, werecarried out as described by Maniatis et at., (1989) Molecular Cloning, ALaboratory Manual; Second edition: Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y., or following the recommended procedures ofmanufacturers of specific products. In most cases enzymes were purchasedfrom New England BioLabs, but other suppliers, and equivalent proceduresmay be used. Oligonucleotide sequences were prepared in an AppliedBiosystems 380A DNA synthesiser from 5′dimethoxytrityl base-protectednucleoside-2-cyanoethyl-N,N′-di-isopropyl-phosphoramidites and protectednucleoside linked to controlled-pore glass supports on a 0.2 μmol scale,according to the protocols supplied by Applied Biosystems Inc.

[0299] Mutants of HCPB, native HCPB and HCPB fusion proteins wereassayed for their ability to convert hippuryl-L-glutamic acid tohippuric acid using an HPLC based assay as described in Example 103 orInternational Patent Application Number WO 96/20011 Example 20.

[0300] Immunoassay techniques were carried out using methods based onthose described by Tijssen, (1985) Practice and Theory of EnzymeImmunoassays, Laboratory Techniques in Biochemistry and MolecularBiology Volume 15, Elsevier Science Publishers, Amsterdam, or followingthe recommended procedures of manufacturers of specific products.

[0301] To generate plasmids capable of expressing the antibody-enzymefusion protein in eukaryotic cells the GS-System (Celltech Biologics)was used (details in International Patent Application Numbers WO87/04462, WO 89/01036, WO 86/05807 and WO 89/10404) with the twoplasmids pEE6 (a derivative of pEE6.hCMV in which the HindIIIrestriction site upstream of the hCMV promoter has been converted to aBglII site {Stephens and Cockett, 1989, Nucleic Acids Research, 17,7110}) and pEE12 (a derivative of pSV2.GS with a number of restrictionsites removed {Bebbington et al, 1992, Bio/Technology, 10. 169}).

[0302] a) Cloning pre-pro-HCPB Up to Restriction Enzyme XmaI Cut Site(Position 1048 in SEQ ID NO: 124)

[0303] Double stranded DNA of plasmid pMF18 (as described inInternational Patent application Number WO 96/20011 Reference Example19), a construct consisting of pre-pro-HCPB cloned into vectorpBluescript II KS+ (Stratagene ), was prepared using standard DNAtechnology (Qiagen plasmid kit or similar), and restriction digestedwith HindIII and XmaI enzymes, being very careful to ensure completedigestion. Restriction enzyme HindIII cuts the pMF18 plasmid just priorto the start of the pre-sequence of the HCPB gene, and XmaI cuts at thecodon for amino acid 240 (proline) of the mature protein, the HindIII toXmaI DNA piece is referred to as the pre-pro-HCPB fragment. DNA of thecorrect size, containing the pre-pro-HCPB fragment (about 1061 basepairs) was purified.

[0304] Double stranded DNA of plasmid vector pUC19 (New England BioLabs)was prepared, restriction digested with HindIII and Xmal, and purified(about 2651 base pairs) in a similar manner to the pre-pro-HCPBfragment. Ligation mixes were prepared to clone the HCPB gene fragmentinto the pUC19 vector, using a molar ratio of about 1 vector to 2.5insert, and a final DNA concentration of about 2.5 ng/μl, in thepresence of T4 DNA ligase. 1 mM ATP and enzyme buffer. Following theligation reaction the DNA mixture was used to transform E. coli strainDH5α. Cell aliquots were plated on L-agar nutrient media containing 100g/ml ampicillin as selection for plasmid vector, and incubated overnightat 37° C. A number of colonies were picked and used formini-preparations of double stranded plasmid DNA. These DNA samples wereanalysed by restriction enzyme digestion, and a construct of the correctconfiguration identified. This plasmid containing the pre-pro-HCPBfragment up to the Xmal site in the mature gene is known as pCF003.

[0305] b) Cloning [A248S,G251T,D253K]HCPB From Position G241+Linker and5 Amino Acids of VH

[0306] To separate the HCPB from the Fd sequence a neutral peptidelinker consisting of (Glycine-Glycine-Glycine-Serine)₃ was introducedinto the sequence during the PCR. In order to generate the fragment ofthe mutant [A248S,G251T,D253K]HCPB sequence (as documented in ReferenceExample 2) and add the peptide linker and the first 5 amino acids of thehumanised 806.077 VH, a PCR was set up using 100 pMols of primers CME00971 and CME 00972 (SEQ ID NOs: 122 and 123) in the presence ofapproximately 5ng of pZen1921 DNA, dNTPs to a final concentration of 200μM, Taq polymerase reaction buffer, and 2.5U of Taq polymerase in afinal volume of 100 μl. The mixture was heated at 94° C. for 10 minutesprior to addition to the Taq enzyme, and the PCR incubation was carriedout using 30 cycles of 94° C. for 1.5 minutes, 55° C. for 2 minutes, and72° C. for 2 minutes, followed by a single incubation of 72° C. for 10minutes at the end of the reaction. The PCR product containing the[A248S,G251T,D253K]HCPB fragment (about 298 base pairs) was analysed forDNA of the correct size by agarose gel electrophoresis and found tocontain predominantly a band of the correct size. The remainder of theproduct from the reaction mix was purified and separated from excessreagents using a microconcentrator column (Centricon™ 100, Amicon),followed by DNA isolation by ethanol/sodium acetate precipitation,centrifugation, vacuum drying and re-suspension in distilled water. Theisolated DNA was restriction digested with enzymes XmaI and EcoRI, and aband of the correct size (about 271 base pairs) purified.

[0307] Double stranded DNA of plasmid pCF003 (described above) preparedusing standard DNA technology (Qiagen plasmid kits or similar), wasrestriction digested with Xmal and EcoRI enzymes, and a band of thecorrect size (about 2696 base pairs) purified.

[0308] Ligation mixes were prepared to clone the mutant HCPB genefragment into the vector, using a molar ratio of about 1 vector to 2.5insert (1 pCF003 to 2.5 [A248S,G251T,D253K]HCPB fragment PCR product),and a final DNA concentration of about 2.5 ng/μl, in the presence of T4DNA ligase, 1 mM ATP and enzyme buffer. Following the ligation reactionthe DNA mixture was used to transform E. coli strain DH5α. Cell aliquotswere plated on L-agar nutrient media containing 100 μg/ml ampicillin asselection for plasmid vector, and incubated overnight at 37° C. About200 colonies were picked and plated onto duplicate sterilenitro-cellulose filters (Schleicher and Schull), pre-wet on plates ofL-agar nutrient media containing 100 μg/ml ampicillin as selection forplasmid vector, and incubated overnight at 37° C. One duplicate platewas stored at 4° C., and acted as a source of live cells for thecolonies, the other plate was treated to denature and fix the DNA fromthe individual colonies to the nitro-cellulose. The nitro-cellulosefilter was removed from the agar plate and placed in succession ontofilter papers (Whatman) soaked in: 1. 10% SDS for 2 minutes; 2. 0.5MNaOH, 1.5M NaCl for 7 minutes; 3. 0.5M NaOH, 1.5M NaCl for 4 minutes; 4.0.5M NaOH, 1.5M NaCl for 2 minutes; 5. 0.5M Tris pH7.4, 1.5M NaCl for 2minutes; and 6. 2×SSC (standard saline citrate) for 2 minutes. Thefilter was then placed on a filter paper (Whatman) soaked in 10×SSC andthe denatured DNA was crossed linked to the nitro-cellulose by ultraviolet light treatment (Spectrolinker XL-1 500 UV crosslinker). Thefilters were allowed to air dry at room temperature, and were thenpre-hybridised at 60° C. for one hour in a solution of 6×SSC with gentleagitation (for example using a Techne HB-1D hybridizer). Notepre-hybridisation blocks non-specific DNA binding sites on the filters.

[0309] In order to determine which colonies contain DNA inserts ofinterest the DNA cross-linked to the nitro-cellulose filter washybridised with a radio-labelled ³²P-DNA probe prepared from the[A248S,G251T,D253K]HCPB purified PCR DNA fragment (see above). About 50ng of DNA was labelled with 50 μCi of ³²P-dCTP (>3000 Ci/mMol) using T7DNA polymerase in a total volume of 50 μl (Pharmacia T7 Quickprime kit),and the reaction allowed to proceed for 15 minutes at 37° C. Thelabelled probe was heated to 95° C. for 2 minutes, to denature thedouble stranded DNA, immediately added to 10 ml of 6×SSC at 60° C., andthis solution was used to replace the pre-hybridisation solution on thefilters. Incubation with gentle agitation was continued for about 3hours at 60° C. After this time the hybridisation solution was drainedoff, and the filters were washed twice at 60° C. in 2×SSC for 15 minuteseach time. Filters were then gently blotted dry, covered with cling film(Saran™ wrap or similar), and exposed against X-ray film (for exampleKodak X-OMAT-ARS™) overnight at room temperature. Following developmentof the film, colonies containing inserts of interest were identified asthose which gave the strongest exposure (darkest spots) on the X-rayfilm. In this series of experiments about 15% of the colonies gavepositive hybridisation. From this 12 colonies were chosen for furtherscreening. These colonies were picked from the duplicate filter,streaked and maintained on L-agar nutrient media containing 100 μg/mlampicillin, and grown in L-broth nutrient media containing 100 μg/mlampicillin.

[0310] The selected colonies were used for mini-preparations of doublestranded plasmid DNA. These DNA samples were analysed by restrictionenzyme digestion, and constructs of the correct configurationidentified. In order to ensure that no changes to the DNA sequence hadbeen introduced during the PCR a number of clones with correctrestriction mapping were taken for DNA preparation using standardtechnology (Qiagen plasmid kits or similar), and the inserts sequencedusing several separate oligonucleotide primers. A construct of thecorrect sequence was identified, and this plasmid containing thepre-pro-[A248S,G251T,D253K]HCPB-linker-humanised 806.077 VH gene up tothe PstI site (at amino acid 5)(position 1301 in SEQ ID NO: 124) istermed pCF004.

[0311] c. Cloning Humanised 806.077 Fd

[0312] Double stranded DNA of plasmid pNG4-VHss-HuVH1-806.077-IgG3CH1′,a construct consisting of the humanised 806.077 version 1 VH with humanIgG3 CH1 and hinge region cloned into vector pNG4 (see Example 44), wasprepared using standard DNA technology (Qiagen plasmid kit or similar),and restriction digested with PstI and XmaI enzymes. DNA of the correctsize, containing the humanised 806.077 Fd fragment (about 854 basepairs) was purified. Double stranded DNA of plasmid vector pUC19 (NewEngland BioLabs) was prepared, restriction digested with PstI and XmaI,and purified (about 2659 base pairs) in a similar manner to thehumanised 806.077 Fd fragment.

[0313] Aliquots of both restricted and purified DNA samples were checkedfor purity and concentration estimation using agarose gelelectrophoresis compared with known standards. From these estimatesligation mixes were prepared to clone the humanised 806.077 Fd genefragment into the pUC19 vector, using a molar ratio of about 1 vector to2.5 insert, and a final DNA concentration of about 2.5 ng/μl, in thepresence of T4 DNA ligase, 1 mM ATP and enzyme buffer.

[0314] Following the ligation reaction the DNA mixture was used totransform E. coli strain DH5α. Cell aliquots were plated on L-agarnutrient media containing 100 μg/ml ampicillin as selection for plasmidvector, and incubated overnight at 37° C. A number of colonies werepicked and used for mini-preparations of double stranded plasmid DNA.These DNA samples were analysed by restriction enzyme digestion, and aconstruct of the correct configuration identified. This plasmidcontaining the humanised 806.077 Fd fragment from the PstI site to theXmal site is known as pCF005.

[0315] d) Cloning Humanised 806.077 Fd into pre-pro-[A248S,G251T,D253K]HCPB-Linker Construct

[0316] Double stranded DNA of plasmid pCF005 (as documented above), wasprepared using standard DNA technology (Qiagen plasmid kit or similar),and restriction digested with PstI and EcoRI enzymes. DNA of the correctsize, containing the humanised 806.077 Fd fragment (about 870 basepairs) was purified. Double stranded DNA of plasmid vector pCF004 (asdocumented above) was prepared, restriction digested with PstI andEcoRI, and purified (about 3950 base pairs) in a similar manner to thehumanised 806.077 Fd fragment. Ligation mixes were prepared to clone thehumanised 806.077 Fd gene fragment into the pCF004 vector, using a molarratio of about 1 vector to 2.5 insert, and a final DNA concentration ofabout 2.5 ng/μl, in the presence of T4 DNA ligase, 1 mM ATP and enzymebuffer.

[0317] Following the ligation reaction, the DNA mixture was used totransform E. coli strain DH5α. Cell aliquots were plated on L-agarnutrient media containing 100 μg/ml ampicillin as selection for plasmidvector, and incubated overnight at 37° C. A number of colonies werepicked and used for mini-preparations of double stranded plasmid DNA.These DNA samples were analysed by restriction enzyme digestion, and aconstruct of the correct configuration identified. This plasmidcontaining the pre-pro-[A248S,G251T,D253K]HCPB-Linker-Fd(humanised806.077) in pUC19 is known as pCF006.

[0318] e) Cloning pre-pro-[A248S, G25 T,D253K]HCPB-linker-(Humanised806. 077)Fd into pEE6 hCMV Vector

[0319] Double stranded DNA of plasmid pCF006 (as documented above), wasprepared using standard DNA technology (Qiagen plasmid kit or similar),and restriction digested with HindIII and EcoRI enzymes. DNA of thecorrect size, containing the fusion protein (about 2185 base pairs) waspurified.

[0320] Double stranded DNA of plasmid vector pEE6 (as documented above)was prepared, restriction digested with HindIII and EcoRI, and purified(about 4775 base pairs) in a similar manner to the fusion protein.Ligation mixes were prepared to clone the humanised 806.077 Fd fusionprotein into the pEE6 vector, using a molar ratio of about 1 vector to2.5 insert, and a final DNA concentration of about 2.5 ng/μl, in thepresence of T4 DNA ligase, 1 mM ATP and enzyme buffer. Following theligation reaction the DNA mixture was used to transform E. coli strainDH5α. Cell aliquots were plated on L-agar nutrient media containing 100μg/ml ampicillin as selection for plasmid vector, and incubatedovernight at 37° C. A number of colonies were picked and used formini-preparations of double stranded plasmid DNA. These DNA samples wereanalysed by restriction enzyme digestion, and a construct of the correctconfiguration identified. This plasmid containing thepre-pro-[A248S,G251T,D253K]HCPB-Linker-Fd(humanised 806.077) in pEE6 isknown as pCF007.

[0321] f) Cloning Humanised 806.077 Light Chain Version 4 into pEE12Vector

[0322] Double stranded DNA of plasmid pNG3-VKss-806.077-HuVK4-HuCK-Neo,a construct consisting of the humanised 806.077 version HuVK4 with humanCK cloned into vector pNG3 (see Examples 12-38), was prepared usingstandard DNA technology (Qiagen plasmid kit or similar), and restrictiondigested with HindIII and EcoRI enzymes. DNA of the correct size,containing the humanised 806.077 light chain (about 2022 base pairs) waspurified. Double stranded DNA of plasmid vector pEE12 was prepared,restriction digested with HindIII and EcoRI, and purified (about 7085base pairs) in a similar manner to the humanised 806.077 light chain.Ligation mixes were prepared to clone the humanised 806.077 light chaininto the pEE12 vector, using a molar ratio of about 1 vector to 2.5insert, and a final DNA concentration of about 2.5 ng/μl, in thepresence of T4 DNA ligase, 1 mM ATP and enzyme buffer. Following theligation reaction the DNA mixture was used to transform E. coli strainDH5α. Cell aliquots were plated on L-agar nutrient media containing 100μg/ml ampicillin as selection for plasmid vector, and incubatedovernight at 37° C. A number of colonies were picked and used formini-preparations of double stranded plasmid DNA. These DNA samples wereanalysed by restriction enzyme digestion, and a construct of the correctconfiguration identified. This plasmid containing the humanised 806.077light chain version 4 is known as pCF008/4.

[0323] g) Cloning CMVp-pre-pro-[A248S,G251T,D253K]HCPB-Linker-(Humanised 806.077)Fd into pCF008/4.

[0324] Double stranded DNA of plasmid pCF007 (as documented above), wasprepared using standard DNA technology (Qiagen plasmid kit or similar),and restriction digested with BglII and SalI enzymes. Restriction enzymeBglII cuts the pCF007 plasmid prior to the start of the CMV MIE leader,promoter and gene for the fusion protein. Restriction enzyme SalI cutsabout 520 base pairs after the stop codons of the mature protein. DNA ofthe correct size, containing the fusion protein (about 4844 base pairs)was purified. Double stranded DNA of plasmid vector pCF008/4 wasprepared, restriction digested with BamHI and SalI, and purified (about7436 base pairs) in a similar manner to the fusion protein. Ligationmixes were prepared to clone the[A248S,G251T,D253K]HCPB-linker-(humanised 806.077)Fd fusion gene intothe pCF008/4 vector, using a molar ratio of about 1 vector to 2.5insert, and a final DNA concentration of about 2.5 ng/μl, in thepresence of T4 DNA ligase, 1 mM ATP and enzyme buffer. Following theligation reaction the DNA mixture was used to transform E. coli strainDH5α. Cell aliquots were plated on L-agar nutrient media containing 100μg/ml ampicillin as selection for plasmid vector, and incubatedovernight at 37° C. A number of colonies were picked and used formini-preparations of double stranded plasmid DNA. These DNA samples wereanalysed by restriction enzyme digestion, and a construct of the correctconfiguration identified. This plasmid containing genes forpro-[A248S,G251T,D253K]HCPB-Linker-F(ab′)₂ (humanised 806.077 antibody)in the GS expression vector pEE12 is known as pCF009 and a plasmid mapis shown in FIG. 2. The DNA and amino acid sequences of the light chainHuVK4 are shown in SEQ ID NOs: 70 and 71. The DNA sequence of thepre-pro-[A248S,G251T,D253K]HCPB-linker-Fd(Humanised 806.077) is shown inSEQ ID NO: 124 and the corresponding amino acid sequence in SEQ ID NO:125.

[0325] h) Expression of Pro-[Mutant]HCPB-Linker-F(ab′)₂(Humanised 806077) From Mouse Myeloma Cells.

[0326] The following method has been used for myeloma expression of all(D253K and G251T,D253K and A248S,G251T,D253K) mutant pro-HCPB enzymefusion proteins. The preferred mouse myeloma cell line is NS0 (Galfreand Milstein, 1981, Methods in Enzymol., 73, 3-46), and is availableform the European Collection of Animal Cell Cultures, PHLS CAMR, PortonDown, Salisbury, Wiltshire, SP4 0JG (ECACC catalogue number 85110503).These cells were grown in Dulbecco's Modified Eagle Medium (DMEM;Gibco/BRL) containing 10% heat inactivated foetal calf serum (FCS).

[0327] For expression ofpro-[A248S,G251T,D253K]HCPB-linker-F(ab′)₂(humanised 806.077) twoplasmids were used, pCF009 (described above) and pRc/RSV (fromInvitrogen, Cats no. V780-20) which contains the neomycin resistancegene for selection of G418 resistant stable cell lines. About 5 μg ofeach plasmid (from 0.5 to 10 μg) were used to transfect approximately8×10⁶ NS0 cells by the method of lipofection (Felgner et al., inMethods: A Companion to Methods in Enzymology, 1993, 5, 67-75) whichinvolves the cationic lipid mediated delivery of polynucleotides intoeukaryotic cells. The cells were harvested by centrifugation, washedwith serum free medium (30 ml), resuspended in 800 μl of medium and keptat 37° C. in a tissue culture flask until the DNA was added. Serum-freemedium (450 μl) was mixed gently with LIPOFECTIN™ reagent (50 μl) andincubated at room temperature for 30 to 45 minutes. This mixture wasadded to 500 μl of medium containing the plasmid DNA mixture (in lessthan 100 μl) and left at room temperature for 15 minutes. Serum freemedium (600 μl) was added to the plasmid DNA-LIPOFECTIN™ mixture, andthe complex added to the cells which were incubated for about 5 hours at37° C. in a CO₂ incubator. The DNA containing medium was then replacedwith normal DMEM medium (8 ml) containing 10% FCS and the cellsincubated overnight. The medium was then again replaced with normal DMEMmedium (8 ml) containing 10% FCS and the cells incubated as previouslywithout selection for 24 hours. At the end of this period the medium waschanged to DMEM containing 10% FCS and G418 selection (1.5 mg/ml), andthe cells diluted (between 1 in 4 and 1 in 20) (approximately 0.5 to1.5×10⁶ cells per plate) in the same medium into micro-titre wells (150μl per well; 2 or more plates per dilution). The micro-titre plates wereincubated for at least two weeks at 37° C. in a CO₂ incubator and thenchecked regularly for viable clone formation.

[0328] Media from wells containing single viable clones was taken fortesting and replaced with fresh media (containing G418). The removedmedia was tested for antibody binding to CEA in an ELISA (in the samemanner as described in International Patent application Number WO96/20011 Reference Example 5 part 1, except that the secondary antibodysolution was changed from anti-mouse to anti-human (goat anti-humanKappa light chain peroxidase conjugate, Sigma A7164). Positive samplesfor the CEA ELISA were also tested for [A248S,G251T,D253K]HCPB enzymeactivity (as described above) following activation (removal of the prodomain from the fusion protein) by trypsin (700 μg/ml in 50 mM Tris-HCland 150 mM NaCl pH 7.6 at 4° C. for 1 hour, the reaction being stoppedby the addition of a five fold excess of soy bean trypsin inhibitor). Anumber of clones were identified which produced media that was positivefor both 806.077 antibody binding to CEA and [A248S,G251T,D253K]HCPBenzyme activity. These were further tested by non-reducing Western blotanalysis (in the same manner as described in International PatentApplication Number WO 96/20011 Reference Example 5 part j, except thatthe antibody solution is changed from anti-mouse to anti-human (goatanti-human Kappa light chain peroxidase conjugate, Sigma A7164) toidentify clones which produce predominately F(ab′)₂(806.077) fusionprotein. These clones were then expanded, tested for stable generationof the fusion protein over a number of generations, and the highestproducers bulked up and stored frozen in liquid nitrogen using standardtechnology.

[0329] Amplification, high-level expression and fermentation of fissionproteins from NS0 myeloma cells was performed in a similar manner tothat described by Bebbington et al. (1992) in Bio/Technology 10,169-175. Fusion protein was purified, and the pro-sequence removed asdescribed in Example 102.

EXAMPLES 76 TO 101 Cloning and Expression of Other Variants ofPro-HCPB-Linker-(Humanised 806.077)Fd+(Humanised 806.077) Light Chain

[0330] The method for the generation of fusion proteins with othermutants of HPCB was similar to that detailed in Example 75 (above), withthe exception that in part b. of Example 75 there was a substitution of[D253K]HCPB or [G251T,D253K]HCPB for [A248S,G251T,D253K]HCPB and theplasmid DNA used in the PCR reaction was pIC11713 (as described inInternational Patent application Number WO 96/20011, Example 15) orpZEN21860 (Reference Example 1) respectively. After cloning,identification, and sequence confirmation the resulting plasmidcontaining pre-pro-[D253K]HCPB-linker orpre-pro-[G251T,D253K]HCPB-linker and humanised 806.077 VH gene up to thePstI site (at amino acid 5) in the pUC19 vector back ground was used inplace of pCF004 in the subsequent cloning reactions.

[0331] The method for generation of fusion proteins with other CH1domains was similar to that detailed in Example 75 (above), with theexception that in part c. of Example 75 there was a substitution ofplasmids containing either humanised 806.077 VH version 1 with humanIgG1 or IgG2 CH1 and hinge regions in place of 806.077-HuVH1-IgG3CH1′(SEQ ID NOs: respectively). After cloning, identification, and sequenceconfirmation the resulting plasmid containing the IgG1 or IgG2 sequencewas used in place of pCF005 in the subsequent actions.

[0332] The method for generation of fusion proteins with other variantsof the humanised 806.077 light chain was similar to that detailed inExample 75 (above), with the exception that in part f. of Example 75there was a substitution of plasmids containing either humanised 806.077Lc version 1 or version 3 in place of 806.077-HuVK4-HuCK (SEQ ID NOs: 51and 96 respectively). After cloning, identification, and sequenceconfirmation the resulting plasmid containing the alternative lightchain sequence was used in place of pCF008/4 in the subsequent cloningreactions. The fusion protein variants for each Example (76 to 101) areshown in the following table. TABLE Ex- am- ple Humanised HumanisedMutant HCPB No. Heavy chain Light chain Enzyme 76 HuVH1-HulgG3HuVK4-HuCk [D253K]HCPB 77 HuVH1-HulgG3 HuVK4-HuCK [G251T,D253K]HCPB 78HuVH1-HulgG3 HuVK1-HuCK [A248S,G251T,D253K]HCPB 79 HuVHI-HulgG3HuVK1-HuCK [D253K]HCPB 80 HuVH1-HulgG3 HuVK1-HuCK [G251T,D253K]HCPB 81HuVH1-HulgG3 HUVK3-HuCK [A248S,G251T,D253K]HCPB 82 HuVH1-HulgG3HuVK3-HuCK [D253K]KCPB 83 HuVH1-HulgG3 HuVK3-HuCK [G251T,D253K]HCPB 84HuVH1-HulgG1 HuVK4-HuCK [A248S,G251T,D253K]HCPB 85 HuVH1-HulgG1HuVK4-HuCK [D253K]HCPB 86 HuVH1-HulgG1 HuVK4-HuCK [G251T,D253K]HCPB 87HuVH1-HulgG1 HuVK1-HuCK [A248S,G251T,D253K]HCPB 88 HuVH1-HulgG1HuVK1-HuCK [D253K]HCPB 89 HuVH1-HulgG1 HuVK1-HuCK [G251T,D253K]HCPB 90HuVH1-HulgG1 HuVK3-HuCK [A248S,G251T,D253K]HCPB 91 HuVH1-HulgG1HuVK3-HuCK [D253K]HCPB 92 HuVH1-HulgG1 HuVK3-HuCK [G251T,D253K]HCPB 93HuVH1-HulgG2 HuVK4-HuCK [A248S,G251T,D253K]HCPB 94 HuVH1-HulgG2HuVK4-HuCK [D253K]HCPB 95 HuVH1-HulgG2 HuVK4-HuCK [G251T,D253K]HCPB 96HuVH1-HulgG2 HuVK1-HuCK [A248S,G251T,D253K]HCPB 97 HuVH1-HulgG2HuVK1-HuCK [D253K]HCPB 98 HuVH1-HulgG2 HuVK1-HuCK [G251T,D253K]KCPB 99HuVH1-HulgG2 HuVK3-HuCK [A248S,G251T,D253K]HCPB 100 HuVH1-HulgG2HuVK3-HuCK [D253K]HCPB 101 HuVH1-HulgG2 HuVK3-HuCK [G251T,D253K]HCPB

EXAMPLE 102 Purification of Proteins Containing 806.077 AntibodySequences

[0333] Purification or enrichment of recombinant F(ab′)₂ orantibody-enzyme fusion proteins may be achieved from myeloma cell, CHOcell or COS cell supernatants by several methods, used either singly ortogether. Purification of murine 806.077 F(ab′)₂, chimeric 806.077F(ab′)₂ constructs and fully humanised 806.077 F(ab′)₂ constructs, andantibody-enzyme fusion protein constructs incorporating these F(ab′)₂constructs were achieved by one or more of several different methods,affinity chromatography or anion exchange chromatography,, or proteinA/protein G chromatography. These techniques can also be applied topurification of 806.077 antibody—B7 fusions (see Example 104).

[0334] a) Antigen Affinity Chromatography

[0335] Carcinoembryonic antigen (CEA), to which the parent murine806.077 antibody was raised, was immobilised on a column (usingPharmacia products). In brief, immobilisation was via a stable esterbond to Sepharose™ High Performance medium NHS-activated prepared incolumns (HiTrap™); coupling of the CEA to the activated matrix wasperformed following the standard instructions provided with the product.

Preparation of a 1 ml Affinity Column

[0336] CEA stock solution (8 mg/ml) was first diluted with couplingbuffer (0.2M sodium hydrogen carbonate, 0.5M sodium chloride; pH8.3) toa final concentration of 0.5 mg/ml. A new column was washed with 6 ml ofice-cold 1 mM HCl at a flow rate not exceeding 1 ml/min. Immediatelyafter, the CEA ligand (1 ml at 0.5 mg/ml) was injected onto the column.The column was sealed at both ends and left to stand for 30 minutes atroom temperature. Excess active groups that had not coupled to theligand were deactivated and any non-specifically bound ligand was washedout of the column by tree rounds of alternating high and low pH washes.The buffers used were 0.5M ethanolamine, 0.5M sodium chloride (pH8.3)and 0.1M sodium acetate, 0.5M sodium chloride (pH 4.0). In each round ofwashes 6 ml of each buffer was washed over the column matrix. Finally,the column was washed into storage buffer (0.05M Na₂HPO₄, 0.1% NaN₃,pH7.0).

Purification Procedure

[0337] The cell culture supernatant containing the desired F(ab′)₂ orfusion construct e.g. chimeric 806.077 F(ab′)₂, humanised 806.077F(ab′)₂, or antibody-enzyme fusion protein was diluted 1:1 withphosphate buffered saline (pH 7.2) and passed over the 1 ml affinitycolumn at a flow rate of 1 ml/min. The column had previously beenequilibrated with phosphate buffered saline (pH7.2; 50 mM sodiumphoshate, 150 mM sodium chloride). The column was washed with 10 columnvolumes of phosphate buffered saline after the cell supernatant hadpassed over it. Bound F(ab′)₂ was eluted with 5 column volumes of 100 mMsodium citrate (pH3.0), with 1 ml fractions of the eluant beingcollected. Detection of the eluted F(ab′)₂ was achieved by Western blotanalysis using a suitable antibody peroxidase conjugate (an anti-humanKappa Light chain -peroxidase conjugate in the case of the fullyhumanised F(ab′)₂, Sigma A-7164) and developing with hydrogen peroxideand 4-chloro-1-naphthol. Appropriate fractions were pooled andconcentrated, using a centrifugal concentrator (Centricon™ 30), wherenecessary.

[0338] b) Anion Exchange Chromatography

[0339] Cell culture supernatant containing the required F(ab′)₂ orfusion construct e.g. chimeric 806.077 F(ab′)₂, humanised 806.077F(ab′)₂, or antibody-enzyme fusion protein was diafiltered into 50 mMTris (using a stirred cell with a 10,000 molecular weight cut-offmembrane) until the ionic strength of the solution was equivilant to thecolumn equilibration buffer. The 40 ml aliquot of the diafilteredsupernatant was loaded on to a suitable column (Pharmacia Mono Q™ 10/10HR) at 2 ml/min. The column was previously equilibrated with 50 mM Tris(pH8.0). Once the supernatant had passed over the column, the column waswashed back to baseline with the equilibration buffer. Bound material onthe column was then eluted with a 0-50% buffer B (50 mM Tris, 1M sodiumchloride pH8.0 ) over 15 column volumes. Elution fractions werecollected (4 ml per fraction ) and those containing the F(ab′)₂ wereidentified by Western blot analysis using a suitable antibody peroxidaseconjugate (an anti-human Kappa Light chain-peroxidase conjugate in thecase of the fully humanised F(ab′)₂, Sigma A-7164) and developing withhydrogen peroxide and 4chloro-1 -naphthol. Appropriate fractions werepooled and concentrated using a centrifugal concentrator (Centricon™30), where necessary.

[0340] c) Protein A and Protein G Purification

[0341] The cell culture supernatant containing the desired F(ab′)₂ orfusion construct (e.g. 806.077 F(ab′)₂, chimeric 806.077 IgG₁ or IgG₂ orIgG₃; pro-HCPB-linker-806.077 F(ab′)₂, 806.077 F(ab′)₂-HCPB) was diluted1:1 with phosphate buffered saline before being loaded on to a columnpreviously equilibrated in phosphate buffered saline (pH7.2). The columnwas washed with phosphate buffered saline, back to baseline, before thebound F(ab′)₂ or fusion protein was eluted with 100 mM sodium citrate(pH 3.0) in the case of the F(ab′)₂ and 50 mM glycine, 100 mM sodiumchloride (pH10.8) in the case of the fusion proteins. Elution fractionswere collected and neutralised by the addition of 125 μl 2M Tris per 1ml of elution volume. Those fractions containing the F(ab′)₂ were pooledand concentrated where necessary using a centrifugal concentrator.

[0342] d) Pro-sequence cleavage:

[0343] For fusion proteins containing a covalently linked pro-sequencee.g.(Pro-HCPB-linker-806.077 F(ab′)₂ ) the pro sequence was cleaved byincubation the fusion with trypsin. This procedure at a milligram (offusion) scale involved the following. Trypsin was mixed with the fusionprotein in a ratio of 1:1000 (trypsin:fusion). The mixture was incubatedfor 24 hours at room temperature (around 22° C.), after which thecleavage of the pro sequence was complete. The fusion protein wasseparated from the pro sequence by recirculating the mixture in one ofthe generic chromatography purification or enrichment protocols.

EXAMPLE 103 Assay of Activity of Antibody-Enzyme Fusion ProteinsContaining Mutant Human CPB Against Hipp-Glu Prodrug Analogues

[0344] Cell culture supernatants or purified antibody-enzyme fusionproteins containing mutants of human CPB (D253K; G252T,D253K;A248S,G251T,D253K: Examples 48-101) are assayed for their ability toconvert hippuryl-L-glutamic acid (Hipp-Glu; Reference Example 9 inInternational Patent Application Number WO 96/20011)) to hippuric acidusing a HPLC based assay.

[0345] The reaction mixture (250 μl) contains either 4 μg of purifiedfusion protein or cell culture supernatant (used either neat or dilutedwith 0.025M Tris-HCl pH7.5; 125 μl) and 0.5 mM Hipp-Glu in 0.025 MTris-HCL, pH 7.5. Samples are incubated for 5 hr at 37° C. The reactionsare terminated by the addition of 250 μl of 30% methanol, 70% phosphatebuffer (50 mM; pH 6.5), 0.2% trifluoroacetic acid and the amount ofhippuric acid generated is quantified by HPLC (using a Hewlett Packard1090 Series 11 with diode array system).

[0346] Samples (50 μl) are injected onto a column (25 cm; HICHROM™Hi-RPB) and separated using a mobile phase of 15% methanol, 85%phosphate buffer (50 mM; pH 6.5) at a flow rate of 1 ml/min. The amountof product (hippuric acid) produced is determined from calibrationcurves generated with known amounts of hippuric acid (Sigma-H6375).Results are expressed as the percentage conversion of substrate intoproduct at 37° C. at times ranging from 30 min-24 h depending on rate ofconversion.

[0347] For antibody-enzyme fusion proteins with an N-terminal proCPB,the pro domain is first removed by treatment with trypsin (700 μg/ml) in50 mM Tris-HCl (pH7.6), 150 mM NaCl at 4° C. for 1 h.

EXAMPLE 104 Preparation of a Human B7.1-humanised 806.077 F(ab′)₂ FusionProtein (hB7-806)

[0348] As in Reference Example 3, a fusion protein consisting of thesignal sequence and extracellular domain of human B7.1 fused directly tothe 5′ coding region of the humanised 806.077 antibody Fd chain isconstructed using PCR techniques. A HindIII-NheI fragment is createdcontaining the natural signal sequence and extracellular domain of humanB7.1 fused to the V_(H) region of a humanised 806.077 antibody heavychain. This is cloned into a suitable vector, for examplepNG4-V_(H)ss-HuIgG²CH1′ or pNG4-VHss-HuIgG3CH1′ (see Examples 39-47)(replacing bases 1-423 in Seq.ID NO: 18), to create a humanB7.1-humanised 806.077 Fd fusion gene. Co-expression of this fusion witha humanised 806.077 L chain (a suitable vector containing the VK4version of humanised 806.077 light chain is pCF008/4; see Example 75) isthen achieved after construction of a co-expression vector usingexpression systems such as those described herein. Such a vector is usedto transfect NS0 myeloma cells and colonies selected on the presence ofCEA binding activity in the culture supernatant. Other humanisedsequences are described in Examples 39-47.

[0349] The hB7-806 fusion protein is expressed from a suitable cell lineand purified using protein-A column as described in Reference Example 3or one of the methods described in Example 102. It should be noted thatpurification methods other than protein-A columns are preferred forhumanised 806.077 antibody fragments and fusion proteins thereof. Thefusion protein can be tested for both antigen and receptor bindingproperties and T-cell co-stimulatory activity when bound to LS174T cellsusing assays set out in Reference Example 3.

EXAMPLE 105 Preparation of Chimeric and Humanised 806.077 F(ab′)₂-CPG2Conjugates

[0350] The procedure described in Example 5 was repeated with the murineF(ab′)₂ protein replaced by one of the chimeric versions described inExample 8 or one of the humanised versions described in Examples 39-47.

EXAMPLE 106 Preparation of Humanised 806.077 Fab-CPG2 Enzyme FusionProtein.

[0351] Humanised 806.077 antibody and bacterial CPG2 enzyme fusionprotein constructs are constructed using PCR methodology similar to thatdescribed for the construction of HuVK4 in Examples 12-38, in whichspecifically designed primers are used in a PCR reaction to amplify theantibody and enzyme gene components (such that the resulting DNAproducts contain overlapping complementary sequence) which are thenjoined via a further “splicing/joining” PCR reaction to make thecomplete antibody-enzyme fusion gene. The fusion protein is created byjoining the 3′ end of Fd humanised 806.077 antibody heavy chain gene tothe 5′ end of the CPG2 structural coding gene to create a Fab-CPG2fusion protein coding gene. In such a construct, the humanised 806.077antibody heavy chain gene component may be terminated after residue K236for the HuVH1-HuIgG1 Fd heavy chain (SEQ ID NO: 93), after residue Val237 for the HuVH1-HuIgG2 Fd heavy chain (SEQ ID NO: 57) or after residueVal 237 heavy chain in the HuVH1-HuIgG3 Fd heavy chain (SEQ ID NO: 95)(thus, in each case, excluding any sequence pertaining to the hingeregion) and may be joined to the first CPG2 residue positionedC-terminal to the signal sequence cleavage site (Minton et al (1984)Gene 31, 31-38). However, in order to obtain optimal antibody bindingand enzymatic properties, it is also envisaged that it may be desirableto incorporate additional residues at the junction between the twoconstituent components.

[0352] The fusion gene is then cloned into a suitable vector, forexample pNG4-VHss-HuIgG2CH1′ (NCIMB no. 40797), after the appropriaterestiction enzyme digestion. isolation of the vector and fusion gene DNAfragment have been made thus replacing the original antibody gene withthat of the fusion protein. Co-expression of the fusion with a humanised806.077 light chain is then achieved after construction of aco-expression vector in a manner analogous to that described in Example11. The co-expression vector is used to transfect NSO myeloma cells andcolonies selected on the presence of CEA and Fd binding activity in theculture supernatant as previously described. The fusion protein can bepurified using a Protein-A column and shown to have both antigen andenzymatic properties using standard test methodology.

EXAMPLE 107 Further Combination of Humanised Heavy and Light ChainVariable Regions Based on Light Chain Sequence VK4

[0353] The procedures described in Examples 12-38 are repeated with thehumanised light chain variable sequence of VK4 (SEQ ID NO: 71) replacedby the modified sequence in which the tyrosine residue (Tyr) at position35 of SEQ ID NO: 71 is replaced by a phenylalanine residue (Phe).

EXAMPLE 108 Further Combination of Humanised Heavy and Light ChainVariable Regions Based on Light Chain Sequence VK4

[0354] The procedures described in Examples 12-38 are repeated with thehumanised light chain variable sequence of VK4 (SEQ ID NO: 71) replacedby the modified sequence in which the phenylalanine residue (Phe atposition 72 of SEQ ID NO: 71 is replaced by a leucine residue (Leu).

EXAMPLE 109 Further Combination of Humanised Heavy and Light ChainVariable Regions Based on Light Chain Sequence VK4

[0355] The procedures described in Examples 12-38 are repeated with thehumanised light chain variable sequence of VK4 (SEQ ID NO: 71) replacedby the modified sequence in which the tyrosine residue (Tyr) at position35 and the phenylalanine residue (Phe) at position 72 of SEQ ID NO: 71are replaced by a phenylalanine residue (Phe) and a leucine residue(Leu) respectively.

EXAMPLE 110 Combination of Humanised Heavy Chain Variable Regions and aChimeric Light Chain Sequence

[0356] The procedures described in Examples 12-38 are repeated with thehumanised light chain variable sequence of replaced by the chimericsequence of SEQ ID NO: 17 described in Example 8.

EXAMPLE 111 -113 Expression of Humanised F(ab′)₂ Fragments with aModified Light Chain VK4 Variable Sequence

[0357] The procedures described in Examples 39-47 are repeated with thevariable light chain sequence described in Example 107 used to make areplacement for the humanised light chain sequence of SEQ ID NO: 99 inwhich the tyrosine residue (Tyr) at position 57 of SEQ ID NO: 99 isreplaced by a phenylalanine residue (Phe).

[0358] Example 111 is the combination of HuVH1-HuIgG1 and the modifiedSEQ ID NO: 99 described above.

[0359] Example 112 is the combination of HuVH1-HuIgG2 and the modifiedSEQ ID NO: 99 described above.

[0360] Example 113 is the combination of HuVH1-HuIgG3 and the modifiedSEQ ID NO: 99 described above.

EXAMPLE 114-116 Expression of Humanised F(ab′)₂ Fragments with aModified Light Chain VK4 Variable Sequence

[0361] The procedures described in Examples 39-47 are repeated with thevariable light chain sequence described in Example 108 used to make areplacement for the humanised light chain sequence of SEQ ID NO: 99 inwhich the phenylalanine residue (Phe) at position 94 of SEQ ID NO: 99 isreplaced by a leucine residue (Leu).

[0362] Example 114 is the combination of HuVH1-HuIgG1 and the modifiedSEQ ID NO: 99 described above.

[0363] Example 115 is the combination of HuVH1-HuIgG2 and the modifiedSEQ ID NO: 99 described above.

[0364] Example 116 is the combination of HuVH1-HuIgG3 and the modifiedSEQ ID NO: 99 described above.

EXAMPLE 117-119 Expression of Humanised F(ab′)₂ Fragments with aModified Light Chain VK4 Variable Sequence

[0365] The procedures described in Examples 39-47 are repeated with thevariable light chain sequence described in Example 109 used to make areplacement for the humanised light chain sequence of SEQ ID NO: 99 inwhich the tyrosine residue (Tyr) at position 57 and the phenylalanineresidue (Phe) at position 94 of SEQ ID NO: 99 is replaced by aphenylalanine residue (Phe) and leucine residue (Leu) respectively.

[0366] Example 117 is the combination of HuVH1-HuIgG1 and the modifiedSEQ ID NO: 99 described above.

[0367] Example 118 is the combination of HuVH1-HuIgG2 and the modifiedSEQ ID NO: 99 described above.

[0368] Example 119 is the combination of HuVH1-HuIgG3 and the modifiedSEQ ID NO: 99 described above.

EXAMPLE 120-122 Expression of Humanised F(ab′)₂ Fragments with aChimeric Light Chain Sequence

[0369] The procedures described in Examples 39-47 are repeated with thechimeric light chain sequence described in Example 1 10 replacing thehumanised light chain sequences used in Examples 39-47

[0370] Example 120 is the combination of HuVH1-HuIgG1 and the chimericlight chain sequence described above.

[0371] Example 121 is the combination of HuVH1-HuIgG2 and the chimericlight chain sequence described above.

[0372] Example 122 is the combination of HuVH1 -HuIgG3 and the chimericlight chain sequence described above.

EXAMPLE 123 Preparation of Humanised Fusion Protein Based on ModifiedLight Chain VK4 Sequence

[0373] The procedures described in Example 48 are repeated but withplasmid pEE14-806.077HuVK4-HuCK replaced by a plasmid containing themodified VK4 sequence of Examples 107 and 111 to 113.

EXAMPLE 124 Preparation of Humanised Fusion Protein Based on ModifiedLight Chain VK4 Sequence

[0374] The procedures described in Example 48 are repeated but withplasmid pEE14-806.077HuVK4-HuCK replaced by a plasmid containing themodified VK4 sequence of Examples 108 and 114 to 116.

EXAMPLE 125 Preparation of Humanised Fusion Protein Based on ModifiedLight Chain VK4 Sequence

[0375] The procedures described in Example 48 are repeated but withplasmid pEE14-806.077HuVK4-HuCK replaced by a plasmid containing themodified VK4 sequence of Examples 109 and 117 to 119.

EXAMPLE 126 Preparation of Humanised Fusion Protein Based on a ChimericLight Chain VK4 Sequence

[0376] The procedures described in Example 48 are repeated but withplasmid pEE14-806.077HuVK4-HuCK replaced by a plasmid containing thechimeric light chain sequence of Examples 110 and 120 to 122.

EXAMPLE 127 Preparation of Humanised Fusion Protein Based on ModifiedLight Chain VK4 Sequence

[0377] The procedures described in Example 75 are repeated but withplasmid pCF008/4 replaced by a plasmid containing the modified VK4sequence of Examples 107 and I1I1 to 113.

EXAMPLE 128 Preparation of Humanised Fusion Protein Based on ModifiedLight Chain VK4 Sequence

[0378] The procedures described in Example 75 are repeated but withplasmid pCF008/4 replaced by a plasmid containing the modified VK4sequence of Examples 108 and 114 to 116.

EXAMPLE 129 Preparation of Humanised Fusion Protein Based on ModifiedLight Chain VK4 Sequence

[0379] The procedures described in Example 75 are repeated but withplasmid pCF008/4 replaced by a plasmid containing the modified VK4sequence of Examples 109 and 117 to 119.

EXAMPLE 130 Preparation of Humanised Fusion Protein Based on a ChimericLight Chain VK4 Sequence

[0380] The procedures described in Example 75 are repeated but withplasmid pCF008/4 replaced by a plasmid containing the chimeric lightchain sequence of Examples 110 and 120 to 122.

REFERENCE EXAMPLE 1 Preparation of Gene Sequence for [G251T,D253K]HCPB

[0381] The method of cloning [G251T,D253K]HCPB in E. coli was verysimilar to the method described in International Patent applicationNumber WO 96/20011, Example 15. Again pICI266 was used as the cloningvector, but the starting material for PCR site directed mutagenesis wasthe [D253K]HCPB gene in plasmid pICI1713 (as described in InternationalPatent Application Number WO 96/20011 Example 15). However, in this casesite directed mutagenesis was used during the PCR amplification of thegene to change the codon at amino acid position 251 in the mature genefrom Glycine to Threonine (GGC to ACT), the G251T change. Also duringthe generation of this mutation a number of other mutations weregenerated at the same (G251) site by using a mixture of oligonucleotideswith codon changes at G251. Individual mutant genes were identifiedfollowing transformation and hybridisation by sequencing across themutation site, prior to complete gene sequencing. In this example onlythe oligonucleotide for introducing the G251T mutation will beconsidered. Two PCR mixtures were prepared, in a manner similar to thatdescribed in International Patent application Number WO 96/20011 Example15. In the first reaction primers were CAN 00402 (SEQ ID NO: 116) andCAN 00734 (SEQ ID NO: 117). In the second reaction primers were CAN00284 (SEQ ID NO: 118) and CAN 01076 (SEQ ID NO: 119). In both reactionsthe starting DNA was pICI1713.

[0382] Aliquots of the two PCR reactions were analysed for DNA of thecorrect size (about 750 and 250 base pairs) and estimation ofconcentration by agarose gel electrophoresis, and found to containpredominantly bands of the correct size. Another PCR was then set upusing each of the first two PCR products, with the two end primers {CAN00402 (SEQ ID NO: 116) and CAN 00284 (SEQ ID NO: 118)}. An aliquot ofthe PCR product was analysed for DNA of the correct size (about 1000base pairs) by agarose gel electrophoresis and found to containpredominantly a band of the correct size. The remainder of the productfrom the reaction mix was purified, the isolated DNA restrictiondigested with enzymes NcoI and EcoRI, and a band of the correct size(about 1000 base pairs) purified in a similar manner to that describedin International Patent application Number WO 96/20011 Example 16.pICI266 double stranded DNA was restriction digested with NcoI and EcoRIenzymes, and DNA of the correct size (about 5600 base pairs) waspurified. Aliquots of both restricted and purified vector and insert DNAsamples were checked for purity and concentration estimation usingagarose gel electrophoresis compared with known standards. From theseestimates ligation mixes were prepared to clone the HCPB gene into thepICI266 vector in a similar manner to that described in InternationalPatent application Number WO 96/20011 Example 16.

[0383] Following the ligation reaction the DNA mixture was used totransform E. coli strain DH5α. colonies were picked and tested byhybridisation. A number of the clones were then taken for plasmid DNApreparation, and were sequenced over the region of PCR mutation in orderto identify clones with the G25 IT change in a manner similar to thatdescribed in International Patent application Number WO 96/20011 Example16. From the sequencing results a clone containing a plasmid with therequired [G251T:D253K]HCPB gene sequence was selected, and the plasmidcalled pZEN1860.

REFERENCE EXAMPLE 2 Preparation of Gene Sequence for[A248S,G251T,D253K]HCPB

[0384] The method of cloning [A248S.G251T,D253K]HCPB in E. coli was verysimilar to the method described in Reference Example 1. The startingmaterial for the PCR site directed mutagenesis was the [G251T,D253K]HCPBgene in plasmid pZEN1860 (described in Reference Example 1) in place ofpICI1713. However, in this case site directed mutagenesis was usedduring the PCR amplification of the gene to change the codon at aminoacid position 248 in the mature gene from alanine to serine (GCT toTTC), the A248S change. Two PCR mixtures were prepared, in a mannersimilar to that described in Reference Examples 1. In the first reactionprimers were CAN 00402 (SEQ ID NO: 116) and CAN 00720 (SEQ ID NO: 120).In the second reaction primers were CAN 00284 (SEQ ID NO: 118) and CAN00726 (SEQ ID NO: 121). In both reactions the starting DNA was pZEN1860.

[0385] Methods of PCR, cloning, expression and identification were thesame as for Reference Example 1. From the sequencing results a clonecontaining a plasmid with the required [A248S,G251T,D253K]HCPB genesequence was selected, and the plasmid called pZEN1921.

REFERENCE EXAMPLE 3 Preparation and Characterisation of a HumanB7.1-Murine A5B7 F(ab′)₂ Fusion Protein (AB7)

[0386] Methods for the preparation, purification and characterisation ofrecombinant murine A5B7 F(ab′)₂ antibody have been published (WO96/20011, Reference Example 5). The cDNA sequence for human B7. 1antigen (also called CD80) has been isolated and described (Freeman G. Jet al, Journal of Immunology, 1989, 143, 2714-2722). In this Example“AB7” refers to human B7.1-murine A5B7 F(ab′)₂ fusion protein and “A5B7”refers to the anti-CEA antibody termed A5B7.

[0387] Using a PCR based strategy we isolated the natural signalsequence and extracellular domain of human B7.1 (encoding amino-acids1-242) from cDNA prepared from cultured Raji cells (ATCC No. CCL 86) andfused it directly upstream from the mature 5′ coding sequence of themurine A5B7 Fd fragment. This involved isolation of the B7.1 sequencewith PCR primers 187/96 and 204/96 (SEQ ID NOS: 126 and 127) and apartial A5B7 Fd sequence with PCR primers 203/96 and 205/96 (SEQ ID NOS:128 and 129). After purification of the PCR products they were mixed inapproximately equimolar amounts and fused by PCR with primers 187/96 and205/96. The resulting PCR product was purified, digested with HindIIIand BstEII (New England Biolabs (UK) Ltd., Wilbury Way, Hitchin, SG4OTY) and cloned into the HindIII-BstEII region of pAF1 using standardprocedures to create the full length human B7.1-murine A5B7 Fd fusion.This fusion gene (SEQ ID NO: 130 - 131) was cloned as a EcoRI-HindIIIfragment into the GS-system™ expression vector pEE6 (Celltech Biologics,Bath Road, Slough, SL1 4 EN) according to the protocols described in WO96/20011, Reference Example 5, to generate vector pAB7.1.

[0388] A BglII-SalI fragment containing the B7.1-A5B7 Fd expressioncassette was then cloned between the BglII and SalI sites of the vectorpAF6 previously described to generate a vector (pAB7.2) capable ofco-expressing the fusion protein and the A5B7 L chain. The vector pAB7.2was then used to transform NSO myeloma cells and colonies selected ontheir ability to grow in the absence of glutamine. Cell lines expressingthe fusion protein were identified by determination of CEA bindingactivity in the culture supernatant using the ELISA described. A cellline expressing suitable levels of fusion protein (1D4) was selected forpurification and characterisation of the AB7 fusion protein.

Purification and Characterisation of the AB7 Fusion Protein

[0389] The secreted recombinant B7.1(35-242)-AB7 F(ab)2, AB7, materialwas purified from culture supernatant using a Protein-A agarose matrixsuch as for example Protein-A Sepharose 4 fast flow as manufactured byPharmacia (Pharmacia Biotech, 23 Grosvenor Rd, St Albans, Herts, AL13AW). The matrix was washed with 2×8 matrix volumes of binding buffer(3M NaCl, 1.5M Glycine, pH 8.9). The culture supernatant containing AB7was diluted 1:1 with the binding buffer. The washed matrix was added tothe diluted culture supernatant (1 ml settled volume of matrix per 40 mlof diluted supernatant) and incubated at 4° C. for 2 hrs with moderateshaking. The matrix was spun down by centrifugation and approx. 75% ofthe supernatant carefully poured off. The matrix was then resuspended inthe residual supernatant and the resulting slurry packed into a column.The column was washed with 5-6 column volumes of 150 mM NaCl, 10 mMNaH₂PO₄, pH7.4. The buffer was then changed to 100 mM NaCitrate pH2.8and elution fractions collected. These fractions were titrated toapproximately pH7.0 by the addition of 2M Tris buffer pH.8.5. Theelution fractions were analysed by non-reducing SDS-PAGE and the peakAB7 fraction(s) retained as the product.

N-Terminal Sequencing

[0390] A sample of AB7 was run on reducing SDS-PAGE and blotted ontoPVDF (polyvinylidene difluoride) membrane (equipment, gels, blottingmembrane and methods from NOVEX, 4202 Sorrento Valley Blvd, San Diego,Calif. 92121, USA.). The protein bands were stained with Coomassie blueand the band at approximately 70 kDa (i.e. B7.1-Fd fusion) wasN-terminally sequenced (Applied Biosystems, 494 Protein Sequencer(Perkin Elmer, ABI division, Kelvin close, Birchwood Science Park North,Warrington, WA3 7PB.) The sequence obtained matched the expectedsequence for mature B7 (ie. after leader sequence cleavage fromamino-acid 35 in SEQ.ID NO: 131, Val Ile His Val etc.).

BIAcore Analysis

[0391] AB7 was analysed using BIAcore surface plasmon resonanceequipment made by Biacore (23 Grosvenor Rd, St. Albans, Herts., AL1 3AW,UK.) according to methods for BIAcore analysis of the CD80/CTLA-4interaction taken from Greene J L, Leytze G M, Emswiler J, Peach R.Bajorath J, Cosand W, and Linsley PS. (1996) J. Biol. Chem. 271,26762-26771. Samples of the purified AB7 product were injected over botha CTLA4-Ig amine coupled surface and a blank (control) amine coupledsurface. Binding could clearly be seen to the CTLA4-Ig surface comparedto the control surface (see FIG. 3). Binding could also be demonstratedbetween CTLA4-Ig and AB7 when the CTLA4-Ig was injected over an aminecoupled AB7 surface.

[0392] Combined with the data from the anti-CEA ELISA these data confirmthat the purified AB7 fusion protein has the biological properties ofboth component parts, namely antigen and receptor binding activities.

Co-stimulatory Activity of the AB7 Fusion Protein

[0393] The ability of the AB7 fusion protein to provide a co-stimulatorysignal to T cells when bound to CEA expressing tumour cells was testedusing an adaptation of a co-stimulation assay format previouslydescribed (Jenkins et al. (1991) J. Immunol. 147:2461). CEA expressingLS174T colo-rectal tumour cells (fixed using 0.5% paraformaldehyde for 5minutes at room temperature) were incubated with 10 μg/ml of the AB7fusion protein (2 hours rotating at 4° .C in RPMI 1640 medium (Gibco.Life Technologies, Paisley, Scotland), containing 0.5% human serum(Sigma AB, Sigma Chemical Co, Dorset, UK.). The cells were washed twiceprior to use and binding of the fusion protein confirmed using afluoroscein isothiocyanate (FITC)-conjugated goat-anti-mouse Ig(Becton-Dickinson UK Ltd, Oxford) and flow cytometry (Facscan, BectonDickinson). To allow the use of unprimed human T cells in the assay, theT cell receptor (TCR) stimulus was provided by an anti T cell receptorantibody (anti-CD3 antibody, OKT-3 Orthoclinical Diagnostics, Amersham,UK) previously coated onto the wells of a 96 well plate. OKT-3 wasimmobilised by incubating purified antibody (2 μg/ml in bicarbonatecoating buffer, pH 9.6 (preformed capsule, Sigma)) overnight at 4° C. in96 well flat bottomed microtitre plates (Costar Corporation, Cambridge,Mass., USA), which were then washed three to four times with PBS.Purified peripheral T cells (from negatively depleted (i.e. pullingout-components other than T cells) from donor human blood using magneticbeads (Dynabeads, Dynal A. S, Oslo, Norway) were added to the wells at2×10⁵/well in 50 μl of RPMI 1640 medium containing 5% human serum. Thefusion protein bound LS174T cells were added to the wells at 5×10⁴/wellin 50 μl of RPMI 1640 medium plus 5% human serum. Finally the volume inall wells was made up to 200 μl using RPMI 1640 medium plus 5% humanserum. Cultures were pulsed with 1.25 μCi of [³H] thymidine (AmershamInternational) after 48 hours and harvested 16 hours later with asemi-automated cell harvester (TomTec harvester, Wallac UK.). Theincorporation of [³H] thymidine into DNA was quantitated using liquidscintillation counting (Betaplate Scint and Betaplate counter, WallacUK.). Data from a typical costimulation assay is displayed in the Tablebelow. TABLE Co-stimulation data αCD3 coated onto wells @ 2 μg/ml (cpm)T cells alone  3582 T cells + αaCD28 28178 T cells + LS174T 12303 Tcells + LS174T + αCD28 25759 T cells + LS174T/fusion protein 41755

[0394] Unprimed T-cells require both T-cell receptor and co-stimulatorysignals. In the assay the T-cell receptor signal is provided by αCD3antibody. Providing co-stimulation via αCD28 (Becton-Dickinson used at0.6 μg/ml) stimulates uptake of [³H] thymidine over 8 fold compared toαCD3 alone. The presence of tumour cells has no significant effect onthis stimulation. Providing the co-stimulatory signal by AB7 fusionprotein bound to tumour cells stimulates uptake of [³H] thymidine bymore than 3 fold over that given by tumour cells alone and over 11 foldhigher than that seen in the absence of co-stimulation. The apparentstimulation provided by tumour cells alone may arise from residualaccessory cells in the purified T-cell population. Similar increases inT cell proliferation were consistently observed in wells containingtumour cell bound fusion protein in each of 5 assays carried outcompared with wells containing T cells and unbound tumour cells.

REFERENCE EXAMPLE 4 Preparation of IgG3-pBSIIKS+

[0395] This example describes the preparation of a vector containing agene for the human IgG3 heavy chain constant and hinge region.

[0396] A gene containing the sequence shown in SEQ ID NO: 115 [thiscontains a sequence (residues 8 to 508) that is similar to SEQ ID NO:25, but with residues 312 and 501 of SEQ ID NO: 25 changed to C and Grespectively], was prepared by PCR by a method similar to that describedby Jayaraman et al. (1991) Proc. Natl. Acad. Sci USA 88, 4084-4088.

[0397] The gene was made in two parts, known as IgG3A and IgG3B. Thesewere cloned separately into the SacI and Xmal sites of pBluescript KS+(Stratagene Cloning Systems) to give vectors IgG3A-pBSIIKS+ clone A7 andIgG3B-pBSIIKS+ clone B17 respectively. IgG3A was made to extend past thePmaCI restriction site (CACGTG at positions 334-339 in SEQ ID NO: 115).Similarly, IgG3B was made such that the 5′ end of the sequence wasupstream of the PmaCI restriction site. To obtain the desired IgG3 genesequence, the intermediate IgG3A and IgG3B vectors were cut with AflIIIand PmaCI. The vector fragment (2823 bp) from IgG3A-pBSIIKS+clone A7,and insert fragment from IgG3B-pBSIIKS+clone B17 (666 bp) were isolatedby electrophoresis in a 1% agarose gel and purified. The fragments wereligated and the ligation mix used to transform E. coli strain DH5α.Clones containing the required gene were identified by digestion ofisolated DNA with SacI and XmaI to give a 520 bp fragment. The sequenceof the insert was confirmed by DNA sequence analysis and clone number F3was designated IgG3-pBSIIKS+.

0 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 131 <210> SEQ ID NO 1<211> LENGTH: 32 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: light chain cDNA foward primer<400> SEQUENCE: 1 ggaagcttga agatggatac agttggtgca gc 32 <210> SEQ ID NO2 <211> LENGTH: 31 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: heavy chain cDNA foward primer<400> SEQUENCE: 2 ggaagcttag acagatgggg gtgtcgtttt g 31 <210> SEQ ID NO3 <211> LENGTH: 34 <212> TYPE: PRT <213> ORGANISM: Mus musculus <400>SEQUENCE: 3 Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Ser GlyAla 1 5 10 15 Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile LysAsp Asn 20 25 30 Tyr Met <210> SEQ ID NO 4 <211> LENGTH: 24 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: light chain cDNA backward primer <400> SEQUENCE: 4gacattcagc tgacccagtc tcca 24 <210> SEQ ID NO 5 <211> LENGTH: 24 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: light chain cDNA backward primer <400> SEQUENCE: 5gacattgagc tcacccagtc tcca 24 <210> SEQ ID NO 6 <211> LENGTH: 22 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: heavy chain cDNA backward primer <400> SEQUENCE: 6aggtsmarct gcagsagtcw gg 22 <210> SEQ ID NO 7 <211> LENGTH: 41 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: heavy chain cDNA backward primer <400> SEQUENCE: 7actagtggaa ttcagtgtga ggtscarctg cagcartcwg g 41 <210> SEQ ID NO 8 <211>LENGTH: 357 <212> TYPE: DNA <213> ORGANISM: Mus musculus <400> SEQUENCE:8 gacattgagc tcacccagtc tccagcaatc atgtctgcat ctccagggga gaaggtcacc 60ataacctgca gtgccagctc aagtgtaact tacatgcact ggttccagca gaagccaggc 120acttctccca aactctggat ttatagcaca tccaacctgg cttctggagt ccctgctcgc 180ttcagtggca gtggatctgg gacctcttac tctctcacaa tcagccgaat ggaggctgaa 240gatgctgcca cttattactg ccagcaaagg agtacttacc cgctcacgtt cggtgctggg 300accaagctgg agctgaaacg ggctgatgct gcaccaactg tatccatctt caagctt 357 <210>SEQ ID NO 9 <211> LENGTH: 108 <212> TYPE: PRT <213> ORGANISM: Musmusculus <400> SEQUENCE: 9 Asp Ile Glu Leu Thr Gln Ser Pro Ala Ile MetSer Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Ile Thr Cys Ser Ala SerSer Ser Val Thr Tyr Met 20 25 30 His Trp Phe Gln Gln Lys Pro Gly Thr SerPro Lys Leu Trp Ile Tyr 35 40 45 Ser Thr Ser Asn Leu Ala Ser Gly Val ProAla Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr IleSer Arg Met Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln GlnArg Ser Thr Tyr Pro Leu Thr 85 90 95 Phe Gly Ala Gly Thr Lys Leu Glu LeuLys Arg Ala 100 105 <210> SEQ ID NO 10 <211> LENGTH: 360 <212> TYPE: DNA<213> ORGANISM: Mus musculus <400> SEQUENCE: 10 gaggtgcagc tgcagcartcwggggcagag cttgtgaggt caggggcctc agtcaagttg 60 tcctgcacag cttctggcttcaacattaaa gacaactata tgcactgggt gaagcagagg 120 cctgaacagg gcctggagtggattgcatgg attgatcctg agaatggtga tactgaatat 180 gccccgaagt tccggggcaaggccactttg actgcagact catcctccaa cacagcctac 240 ctgcacctca gcagcctgacatctgaggac actgccgtct attactgtca tgtcctgatc 300 tatgctggtt atttggctatggactactgg ggtcaaggaa cctcagtcgc cgtctcctca 360 <210> SEQ ID NO 11 <211>LENGTH: 120 <212> TYPE: PRT <213> ORGANISM: Mus musculus <400> SEQUENCE:11 Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Ser Gly Ala 1 510 15 Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Asn 2025 30 Tyr Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile 3540 45 Ala Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala Pro Lys Phe 5055 60 Arg Gly Lys Ala Thr Leu Thr Ala Asp Ser Ser Ser Asn Thr Ala Tyr 6570 75 80 Leu His Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys85 90 95 His Val Leu Ile Tyr Ala Gly Tyr Leu Ala Met Asp Tyr Trp Gly Gln100 105 110 Gly Thr Ser Val Ala Val Ser Ser 115 120 <210> SEQ ID NO 12<211> LENGTH: 39 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: light chain primer <400>SEQUENCE: 12 aagctttccc gcggggacat tgagctcacc cagtctcca 39 <210> SEQ IDNO 13 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: light chain primer<400> SEQUENCE: 13 aagcttctcg agcttggtcc cagcaccgaa 30 <210> SEQ ID NO14 <211> LENGTH: 36 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: heavy chain primer <400>SEQUENCE: 14 aagcttggaa ttcagtgtga ggtgcagctg cagcag 36 <210> SEQ ID NO15 <211> LENGTH: 33 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: heavy chain primer <400>SEQUENCE: 15 aagcttcgag ctcacggcga ctgaggttcc ttg 33 <210> SEQ ID NO 16<211> LENGTH: 705 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: chimaeric light chain sequence<400> SEQUENCE: 16 atggattttc aagtgcagat tttcagcttc ctgctaatcagtgcttcagt cataatgtcc 60 cgcggggaca ttgagctcac ccagtctcca gcaatcatgtctgcatctcc aggggagaag 120 gtcaccataa cctgcagtgc cagctcaagt gtaacttacatgcactggtt ccagcagaag 180 ccaggcactt ctcccaaact ctggatttat agcacatccaacctggcttc tggagtccct 240 gctcgcttca gtggcagtgg atctgggacc tcttactctctcacaatcag ccgaatggag 300 gctgaagatg ctgccactta ttactgccag caaaggagtacttacccgct cacgttcggt 360 gctgggacca agctcgagat caaacggact gtggctgcaccatctgtctt catcttcccg 420 ccatctgatg agcagttgaa atctggaact gcctctgttgtgtgcctgct gaataacttc 480 tatcccagag aggccaaagt acagtggaag gtggataacgccctccaatc gggtaactcc 540 caggagagtg tcacagagca ggacagcaag gacagcacctacagcctcag cagcaccctg 600 acgctgagca aagcagacta cgagaaacac aaagtctacgcctgcgaagt cacccatcag 660 ggcctgagtt cgcccgtcac aaagagcttc aacaggggagagtgt 705 <210> SEQ ID NO 17 <211> LENGTH: 235 <212> TYPE: PRT <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:chimaeric light chain sequence <400> SEQUENCE: 17 Met Asp Phe Gln ValGln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser 1 5 10 15 Val Ile Met SerArg Gly Asp Ile Glu Leu Thr Gln Ser Pro Ala Ile 20 25 30 Met Ser Ala SerPro Gly Glu Lys Val Thr Ile Thr Cys Ser Ala Ser 35 40 45 Ser Ser Val ThrTyr Met His Trp Phe Gln Gln Lys Pro Gly Thr Ser 50 55 60 Pro Lys Leu TrpIle Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro 65 70 75 80 Ala Arg PheSer Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile 85 90 95 Ser Arg MetGlu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Arg 100 105 110 Ser ThrTyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys 115 120 125 ArgThr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 130 135 140Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 145 150155 160 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln165 170 175 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys AspSer 180 185 190 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala AspTyr Glu 195 200 205 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln GlyLeu Ser Ser 210 215 220 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225230 235 <210> SEQ ID NO 18 <211> LENGTH: 765 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:chimaeric HuIgG2 Fd construct <400> SEQUENCE: 18 atgaagttgt ggctgaactggattttcctt gtaacacttt taaatggaat tcagtgtgag 60 gtgcagctgc agcartcaggggcagagctt gtgaggtcag gggcctcagt caagttgtcc 120 tgcacagctt ctggcttcaacattaaagac aactatatgc actgggtgaa gcagaggcct 180 gaacagggcc tggagtggattgcatggatt gatcctgaga atggtgatac tgaatatgcc 240 ccgaagttcc ggggcaaggccactttgact gcagactcat cctccaacac agcctacctg 300 cacctcagca gcctgacatctgaggacact gccgtctatt actgtcatgt cctgatctat 360 gctggttatt tggctatggactactggggt caaggaacct cagtcgccgt gagctcggct 420 agcaccaagg gaccatcggtcttccccctg gccccctgct ccaggagcac ctccgagagc 480 acagccgccc tgggctgcctggtcaaggac tacttccccg aaccggtgac ggtgtcgtgg 540 aactcaggcg ctctgaccagcggcgtgcac accttcccgg ctgtcctaca gtcctcagga 600 ctctactccc tcagcagcgtcgtgacggtg ccctccagca acttcggcac ccagacctac 660 acctgcaacg tagatcacaagcccagcaac accaaggtgg acaagacagt tgagcgcaaa 720 tgttgtgtcg agtgcccaccgtgcccggcg ccacctgtgg ccggc 765 <210> SEQ ID NO 19 <211> LENGTH: 255<212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: chimaeric HuIgG2 Fd construct <400> SEQUENCE: 19 MetLys Leu Trp Leu Asn Trp Ile Phe Leu Val Thr Leu Leu Asn Gly 1 5 10 15Ile Gln Cys Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg 20 25 30Ser Gly Ala Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile 35 40 45Lys Asp Asn Tyr Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu 50 55 60Glu Trp Ile Ala Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala 65 70 7580 Pro Lys Phe Arg Gly Lys Ala Thr Leu Thr Ala Asp Ser Ser Ser Asn 85 9095 Thr Ala Tyr Leu His Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val 100105 110 Tyr Tyr Cys His Val Leu Ile Tyr Ala Gly Tyr Leu Ala Met Asp Tyr115 120 125 Trp Gly Gln Gly Thr Ser Val Ala Val Ser Ser Ala Ser Thr LysGly 130 135 140 Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr SerGlu Ser 145 150 155 160 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr PhePro Glu Pro Val 165 170 175 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr SerGly Val His Thr Phe 180 185 190 Pro Ala Val Leu Gln Ser Ser Gly Leu TyrSer Leu Ser Ser Val Val 195 200 205 Thr Val Pro Ser Ser Asn Phe Gly ThrGln Thr Tyr Thr Cys Asn Val 210 215 220 Asp His Lys Pro Ser Asn Thr LysVal Asp Lys Thr Val Glu Arg Lys 225 230 235 240 Cys Cys Val Glu Cys ProPro Cys Pro Ala Pro Pro Val Ala Gly 245 250 255 <210> SEQ ID NO 20 <211>LENGTH: 121 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: chimaeric HuIgG1CH1′ Fd construct<400> SEQUENCE: 20 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala ProSer Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys LeuVal Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser GlyAla Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser SerGly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser LeuGly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Asn Pro Ser AsnThr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr HisThr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro 115120 <210> SEQ ID NO 21 <211> LENGTH: 369 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: chimaericHuIgG1CH1′ Fd construct <400> SEQUENCE: 21 gcctccacca agggcccatcggtcttcccc ctggcaccct cctccaagag cacctctggg 60 ggcacagcgg ccctgggctgcctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120 tggaactcag gcgccctgaccagcggcgtg cacaccttcc cggctgtcct acagtcctca 180 ggactctact ccctcagcagcgtggtgact gtgccctcca gcagcttggg cacccagacc 240 tacatctgca acgtgaatcacaaccccagc aacaccaagg tcgacaagaa agttgagccc 300 aaatcttgtg acaagacgcacacgtgcccg ccgtgcccgg ctccggaact gctgggtggc 360 ccgtaatag 369 <210> SEQID NO 22 <211> LENGTH: 116 <212> TYPE: PRT <213> ORGANISM: Homo sapiens<400> SEQUENCE: 22 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala ProCys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys LeuVal Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser GlyAla Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser SerGly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn PheGly Thr Gln Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser AsnThr Lys Val Asp Lys 85 90 95 Thr Val Glu Arg Lys Cys Cys Val Glu Cys ProPro Cys Pro Ala Pro 100 105 110 Pro Val Ala Gly 115 <210> SEQ ID NO 23<211> LENGTH: 348 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400>SEQUENCE: 23 gctagcacca agggaccatc ggtcttcccc ctggccccct gctccaggagcacctccgag 60 agcacagccg ccctgggctg cctggtcaag gactacttcc ccgaaccggtgacggtgtcg 120 tggaactcag gcgctctgac cagcggcgtg cacaccttcc cggctgtcctacagtcctca 180 ggactctact ccctcagcag cgtcgtgacg gtgccctcca gcaacttcggcacccagacc 240 tacacctgca acgtagatca caagcccagc aacaccaagg tggacaagacagttgagcgc 300 aaatgttgtg tcgagtgccc accgtgcccg gcgccacctg tggccggc 348<210> SEQ ID NO 24 <211> LENGTH: 167 <212> TYPE: PRT <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: chimaericHuIgG3CH1′ Fd construct <400> SEQUENCE: 24 Ala Ser Thr Lys Gly Pro SerVal Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Gly Gly ThrAla Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val ThrVal Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe ProAla Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val ThrVal Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Thr Cys Asn ValAsn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Leu LysThr Pro Leu Gly Asp Thr Thr His Thr Cys Pro 100 105 110 Arg Cys Pro GluPro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg 115 120 125 Cys Pro GluPro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys 130 135 140 Pro GluPro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro 145 150 155 160Ala Pro Glu Leu Leu Gly Gly 165 <210> SEQ ID NO 25 <211> LENGTH: 501<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: chimaeric HuIgG3CH1′ Fd construct <400> SEQUENCE: 25gctagcacca agggcccatc ggtcttcccc ctggcgccct gctccaggag cacctctggg 60ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 240tacacctgca acgtgaatca caagcccagc aacaccaagg tggacaagag agtggagctg 300aaaaccccac ttggtgacac aactcacacg tgccctaggt gtcctgaacc taaatcttgt 360gacacacctc ccccgtgccc acggtgccca gagcccaaat cttgcgacac gcccccaccg 420tgtcccagat gtcctgaacc aaagagctgt gacactccac cgccctgccc gaggtgccca 480gcacctgaac tcctgggagg a 501 <210> SEQ ID NO 26 <211> LENGTH: 10 <212>TYPE: PRT <213> ORGANISM: Mus musculus <400> SEQUENCE: 26 Ser Ala SerSer Ser Val Thr Tyr Met His 1 5 10 <210> SEQ ID NO 27 <211> LENGTH: 7<212> TYPE: PRT <213> ORGANISM: Mus musculus <400> SEQUENCE: 27 Ser ThrSer Asn Leu Ala Ser 1 5 <210> SEQ ID NO 28 <211> LENGTH: 9 <212> TYPE:PRT <213> ORGANISM: Mus musculus <400> SEQUENCE: 28 Gln Gln Arg Ser ThrTyr Pro Leu Thr 1 5 <210> SEQ ID NO 29 <211> LENGTH: 5 <212> TYPE: PRT<213> ORGANISM: Mus musculus <400> SEQUENCE: 29 Asp Asn Tyr Met His 1 5<210> SEQ ID NO 30 <211> LENGTH: 9 <212> TYPE: PRT <213> ORGANISM: Musmusculus <400> SEQUENCE: 30 Phe Asn Ile Lys Asp Asn Tyr Met His 1 5<210> SEQ ID NO 31 <211> LENGTH: 17 <212> TYPE: PRT <213> ORGANISM: Musmusculus <400> SEQUENCE: 31 Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu TyrAla Pro Lys Phe Arg 1 5 10 15 Gly <210> SEQ ID NO 32 <211> LENGTH: 11<212> TYPE: PRT <213> ORGANISM: Mus musculus <400> SEQUENCE: 32 Leu IleTyr Ala Gly Tyr Leu Ala Met Asp Tyr 1 5 10 <210> SEQ ID NO 33 <211>LENGTH: 13 <212> TYPE: PRT <213> ORGANISM: Mus musculus <400> SEQUENCE:33 His Val Leu Ile Tyr Ala Gly Tyr Leu Ala Met Asp Tyr 1 5 10 <210> SEQID NO 34 <211> LENGTH: 60 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: polylinker sequence<400> SEQUENCE: 34 tcgagagatc taagcttccg cgggaattcc tcgaggagctccccggggga tccgtcgact 60 <210> SEQ ID NO 35 <211> LENGTH: 60 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: polylinker sequence <400> SEQUENCE: 35 ctagagtcgacggatccccc ggggagctcc tcgaggaatt cccgcggaag cttagatctc 60 <210> SEQ IDNO 36 <211> LENGTH: 29 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: polyA region PCR primer<400> SEQUENCE: 36 aagcttcccg ggtattaaag cagaacttg 29 <210> SEQ ID NO 37<211> LENGTH: 29 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: polyA region PCR primer <400>SEQUENCE: 37 actagtggat cccagacatg ataagatac 29 <210> SEQ ID NO 38 <211>LENGTH: 39 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: mutagenesis PCR primer <400> SEQUENCE:38 ggtctatata agcagagctg tctggctaac tagagaacc 39 <210> SEQ ID NO 39<211> LENGTH: 39 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: mutagenesis PCR primer <400>SEQUENCE: 39 ggttctctag ttagccagac agctctgctt atatagacc 39 <210> SEQ IDNO 40 <211> LENGTH: 19 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: flanking PCR primer<400> SEQUENCE: 40 ggactttcct acttggcag 19 <210> SEQ ID NO 41 <211>LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: flanking PCR primer <400> SEQUENCE: 41ggcaactaga aggcacagtc 20 <210> SEQ ID NO 42 <211> LENGTH: 77 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Kozak recognition and light chain signal sequences <400>SEQUENCE: 42 agcttgccgc caccatggat tttcaagtgc agattttcag cttcctgctaatcagtgctt 60 cagtcataat gtcccgc 77 <210> SEQ ID NO 43 <211> LENGTH: 71<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Kozak recognition and light chain signal sequences<400> SEQUENCE: 43 gggacattat gactgaagca ctgattagca ggaagctgaaaatctgcact tgaaaatcca 60 tggtggcggc a 71 <210> SEQ ID NO 44 <211>LENGTH: 61 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Kozak recognition and heavy chainsignal sequences <400> SEQUENCE: 44 agcttgccgc caccatgaag ttgtggctgaactggatttt ccttgtaaca cttttaaatg 60 g 61 <210> SEQ ID NO 45 <211>LENGTH: 61 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: 223> OTHER INFORMATION: Kozak recognition and heavy chainsignal sequences <400> SEQUENCE: 45 aattccattt aaaagtgtta caaggaaaatccagttcagc cacaacttca tggtggcggc 60 a 61 <210> SEQ ID NO 46 <211>LENGTH: 357 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: human light chain kappa constantregion insert <400> SEQUENCE: 46 aagcttctcg agatcaaacg gactgtggctgcaccatctg tcttcatctt cccgccatct 60 gatgagcagt tgaaatctgg aactgcctctgttgtgtgcc tgctgaataa cttctatccc 120 agagaggcca aagtacagtg gaaggtggataacgccctcc aatcgggtaa ctcccaggag 180 agtgtcacag agcaggacag caaggacagcacctacagcc tcagcagcac cctgacgctg 240 agcaaagcag actacgagaa acacaaagtctacgcctgcg aagtcaccca tcagggcctg 300 agttcgcccg tcacaaagag cttcaacaggggagagtgtt aatagcccgg gactagt 357 <210> SEQ ID NO 47 <211> LENGTH: 381<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: human heavy chain 1gG2CH1 constant region insert<400> SEQUENCE: 47 ggaagcttga gctcggctag caccaaggga ccatcggtcttccccctggc cccctgctcc 60 aggagcacct ccgagagcac agccgccctg ggctgcctggtcaaggacta cttccccgaa 120 ccggtgacgg tgtcgtggaa ctcaggcgct ctgaccagcggcgtgcacac cttcccggct 180 gtcctacagt cctcaggact ctactccctc agcagcgtcgtgacggtgcc ctccagcaac 240 ttcggcaccc agacctacac ctgcaacgta gatcacaagcccagcaacac caaggtggac 300 aagacagttg agcgcaaatg ttgtgtcgag tgcccaccgtgcccggcgcc acctgtggcc 360 ggctaatagc ccgggactag t 381 <210> SEQ ID NO 48<211> LENGTH: 342 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: humanised antibody variableregion <400> SEQUENCE: 48 aagctttccc gcggcgacat ccagatgacc cagagcccaagcagcctgag cgctagcgtg 60 ggtgacagag tgaccatcac gtgtagtgcc agctcaagtgtaacttacat gcactggtac 120 cagcagaagc caggtaaggc tccaaagctg ctgatctacagcacatccaa cctggcttct 180 ggtgtgccaa gcagattctc cggaagcggt agcggcaccgactacacctt caccatcagc 240 agcctccagc cagaggatat cgccacctac tactgccagcagaggagtac ttacccgctc 300 acgttcggcc aagggaccaa gctcgagatc aaacggacta gt342 <210> SEQ ID NO 49 <211> LENGTH: 321 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: humanizedlight chain variable region <400> SEQUENCE: 49 gacatccaga tgacccagagcccaagcagc ctgagcgcta gcgtgggtga cagagtgacc 60 atcacgtgta gtgccagctcaagtgtaact tacatgcact ggtaccagca gaagccaggt 120 aaggctccaa agctgctgatctacagcaca tccaacctgg cttctggtgt gccaagcaga 180 ttctccggaa gcggtagcggcaccgactac accttcacca tcagcagcct ccagccagag 240 gatatcgcca cctactactgccagcagagg agtacttacc cgctcacgtt cggccaaggg 300 accaagctcg agatcaaacg g321 <210> SEQ ID NO 50 <211> LENGTH: 107 <212> TYPE: PRT <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: humanizedlight chain variable region <400> SEQUENCE: 50 Asp Ile Gln Met Thr GlnSer Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr IleThr Cys Ser Ala Ser Ser Ser Val Thr Tyr Met 20 25 30 His Trp Tyr Gln GlnLys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 35 40 45 Ser Thr Ser Asn LeuAla Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr AspTyr Thr Phe Thr Ile Ser Ser Leu Gln Pro Glu 65 70 75 80 Asp Ile Ala ThrTyr Tyr Cys Gln Gln Arg Ser Thr Tyr Pro Leu Thr 85 90 95 Phe Gly Gln GlyThr Lys Leu Glu Ile Lys Arg 100 105 <210> SEQ ID NO 51 <211> LENGTH: 705<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: complete humanised light chain sequence <400>SEQUENCE: 51 atggattttc aagtgcagat tttcagcttc ctgctaatca gtgcttcagtcataatgtcc 60 cgcggcgaca tccagatgac ccagagccca agcagcctga gcgctagcgtgggtgacaga 120 gtgaccatca cgtgtagtgc cagctcaagt gtaacttaca tgcactggtaccagcagaag 180 ccaggtaagg ctccaaagct gctgatctac agcacatcca acctggcttctggtgtgcca 240 agcagattct ccggaagcgg tagcggcacc gactacacct tcaccatcagcagcctccag 300 ccagaggata tcgccaccta ctactgccag cagaggagta cttacccgctcacgttcggc 360 caagggacca agctcgagat caaacggact gtggctgcac catctgtcttcatcttcccg 420 ccatctgatg agcagttgaa atctggaact gcctctgttg tgtgcctgctgaataacttc 480 tatcccagag aggccaaagt acagtggaag gtggataacg ccctccaatcgggtaactcc 540 caggagagtg tcacagagca ggacagcaag gacagcacct acagcctcagcagcaccctg 600 acgctgagca aagcagacta cgagaaacac aaagtctacg cctgcgaagtcacccatcag 660 ggcctgagtt cgcccgtcac aaagagcttc aacaggggag agtgt 705<210> SEQ ID NO 52 <211> LENGTH: 235 <212> TYPE: PRT <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: completehumanised light chain sequence <400> SEQUENCE: 52 Met Asp Phe Gln ValGln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser 1 5 10 15 Val Ile Met SerArg Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser 20 25 30 Leu Ser Ala SerVal Gly Asp Arg Val Thr Ile Thr Cys Ser Ala Ser 35 40 45 Ser Ser Val ThrTyr Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala 50 55 60 Pro Lys Leu LeuIle Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro 65 70 75 80 Ser Arg PheSer Gly Ser Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile 85 90 95 Ser Ser LeuGln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Arg 100 105 110 Ser ThrTyr Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 115 120 125 ArgThr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 130 135 140Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 145 150155 160 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln165 170 175 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys AspSer 180 185 190 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala AspTyr Glu 195 200 205 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln GlyLeu Ser Ser 210 215 220 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225230 235 <210> SEQ ID NO 53 <211> LENGTH: 385 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:humanised heavy chain PCR fragment <400> SEQUENCE: 53 gaagcttggaattcagtgtg aggtgcagct gcagcagagc ggtccaggtc tcgtacggcc 60 tagccagaccctgagcctca cgtgcaccgc atctggcttc aacattaagg acaattacat 120 gcactgggtgagacagccac ctggacgagg ccttgagtgg attggatgga ttgaccctga 180 gaatggtgacactgagtacg cacctaagtt tcgcggccgc gtgacaatgc tggcagacac 240 tagtaagaaccagttcagcc tgagactcag cagcgtgaca gccgccgaca ccgcggtcta 300 ttattgtcacgtcctgatat acgccgggta tctggcaatg gactactggg gccaagggac 360 cctcgtcaccgtgagctcga ctagt 385 <210> SEQ ID NO 54 <211> LENGTH: 360 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: humanised antibody variable region <400> SEQUENCE: 54gaggtgcagc tgcagcagag cggtccaggt ctcgtacggc ctagccagac cctgagcctc 60acgtgcaccg catctggctt caacattaag gacaattaca tgcactgggt gagacagcca 120cctggacgag gccttgagtg gattggatgg attgaccctg agaatggtga cactgagtac 180gcacctaagt ttcgcggccg cgtgacaatg ctggcagaca ctagtaagaa ccagttcagc 240ctgagactca gcagcgtgac agccgccgac accgcggtct attattgtca cgtcctgata 300tacgccgggt atctggcaat ggactactgg ggccaaggga ccctcgtcac cgtgagctcg 360<210> SEQ ID NO 55 <211> LENGTH: 120 <212> TYPE: PRT <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: humanisedantibody variable region <400> SEQUENCE: 55 Glu Val Gln Leu Gln Gln SerGly Pro Gly Leu Val Arg Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr CysThr Ala Ser Gly Phe Asn Ile Lys Asp Asn 20 25 30 Tyr Met His Trp Val ArgGln Pro Pro Gly Arg Gly Leu Glu Trp Ile 35 40 45 Gly Trp Ile Asp Pro GluAsn Gly Asp Thr Glu Tyr Ala Pro Lys Phe 50 55 60 Arg Gly Arg Val Thr MetLeu Ala Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Arg Leu Ser SerVal Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 His Val Leu Ile TyrAla Gly Tyr Leu Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu ValThr Val Ser Ser 115 120 <210> SEQ ID NO 56 <211> LENGTH: 765 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: complete humanised Fd heavy chain sequence <400> SEQUENCE:56 atgaagttgt ggctgaactg gattttcctt gtaacacttt taaatggaat tcagtgtgag 60gtgcagctgc agcagagcgg tccaggtctc gtacggccta gccagaccct gagcctcacg 120tgcaccgcat ctggcttcaa cattaaggac aattacatgc actgggtgag acagccacct 180ggacgaggcc ttgagtggat tggatggatt gaccctgaga atggtgacac tgagtacgca 240cctaagtttc gcggccgcgt gacaatgctg gcagacacta gtaagaacca gttcagcctg 300agactcagca gcgtgacagc cgccgacacc gcggtctatt attgtcacgt cctgatatac 360gccgggtatc tggcaatgga ctactggggc caagggaccc tcgtcaccgt gagctcggct 420agcaccaagg gaccatcggt cttccccctg gccccctgct ccaggagcac ctccgagagc 480acagccgccc tgggctgcct ggtcaaggac tacttccccg aaccggtgac ggtgtcgtgg 540aactcaggcg ctctgaccag cggcgtgcac accttcccgg ctgtcctaca gtcctcagga 600ctctactccc tcagcagcgt cgtgacggtg ccctccagca acttcggcac ccagacctac 660acctgcaacg tagatcacaa gcccagcaac accaaggtgg acaagacagt tgagcgcaaa 720tgttgtgtcg agtgcccacc gtgcccggcg ccacctgtgg ccggc 765 <210> SEQ ID NO 57<211> LENGTH: 255 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: complete humanised Fd heavychain sequence <400> SEQUENCE: 57 Met Lys Leu Trp Leu Asn Trp Ile PheLeu Val Thr Leu Leu Asn Gly 1 5 10 15 Ile Gln Cys Glu Val Gln Leu GlnGln Ser Gly Pro Gly Leu Val Arg 20 25 30 Pro Ser Gln Thr Leu Ser Leu ThrCys Thr Ala Ser Gly Phe Asn Ile 35 40 45 Lys Asp Asn Tyr Met His Trp ValArg Gln Pro Pro Gly Arg Gly Leu 50 55 60 Glu Trp Ile Gly Trp Ile Asp ProGlu Asn Gly Asp Thr Glu Tyr Ala 65 70 75 80 Pro Lys Phe Arg Gly Arg ValThr Met Leu Ala Asp Thr Ser Lys Asn 85 90 95 Gln Phe Ser Leu Arg Leu SerSer Val Thr Ala Ala Asp Thr Ala Val 100 105 110 Tyr Tyr Cys His Val LeuIle Tyr Ala Gly Tyr Leu Ala Met Asp Tyr 115 120 125 Trp Gly Gln Gly ThrLeu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 130 135 140 Pro Ser Val PhePro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser 145 150 155 160 Thr AlaAla Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 165 170 175 ThrVal Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 180 185 190Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 195 200205 Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val 210215 220 Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys225 230 235 240 Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val AlaGly 245 250 255 <210> SEQ ID NO 58 <211> LENGTH: 40 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: humanised light chain variable region variant insert <400>SEQUENCE: 58 ggcgacatcc agctgaccca gagcccaagc agcctgagcg 40 <210> SEQ IDNO 59 <211> LENGTH: 46 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: humanised light chainvariable region variant insert <400> SEQUENCE: 59 ctagcgctca ggctgcttgggctctgggtc agctggatgt cgccgc 46 <210> SEQ ID NO 60 <211> LENGTH: 321<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: humanised light chain variable region variant <400>SEQUENCE: 60 gacatccagc tgacccagag cccaagcagc ctgagcgcta gcgtgggtgacagagtgacc 60 atcacgtgta gtgccagctc aagtgtaact tacatgcact ggtaccagcagaagccaggt 120 aaggctccaa agctgctgat ctacagcaca tccaacctgg cttctggtgtgccaagcaga 180 ttctccggaa gcggtagcgg caccgactac accttcacca tcagcagcctccagccagag 240 gatatcgcca cctactactg ccagcagagg agtacttacc cgctcacgttcggccaaggg 300 accaagctcg agatcaaacg g 321 <210> SEQ ID NO 61 <211>LENGTH: 107 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: humanised light chain variable regionvariant <400> SEQUENCE: 61 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser LeuSer Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Ser Ala SerSer Ser Val Thr Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Lys AlaPro Lys Leu Leu Ile Tyr 35 40 45 Ser Thr Ser Asn Leu Ala Ser Gly Val ProSer Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Tyr Thr Phe Thr IleSer Ser Leu Gln Pro Glu 65 70 75 80 Asp Ile Ala Thr Tyr Tyr Cys Gln GlnArg Ser Thr Tyr Pro Leu Thr 85 90 95 Phe Gly Gln Gly Thr Lys Leu Glu IleLys Arg 100 105 <210> SEQ ID NO 62 <211> LENGTH: 40 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: humanised light chain variable region variant <400>SEQUENCE: 62 ggccagatcg tgctgaccca gagcccaagc agcctgagcg 40 <210> SEQ IDNO 63 <211> LENGTH: 46 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: humanised light chainvariable region variant <400> SEQUENCE: 63 ctagcgctca ggctgcttgggctctgggtc agcacgatct ggccgc 46 <210> SEQ ID NO 64 <211> LENGTH: 321<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: humanised light chain variable region variant <400>SEQUENCE: 64 cagatcgtgc tgacccagag cccaagcagc ctgagcgcta gcgtgggtgacagagtgacc 60 atcacgtgta gtgccagctc aagtgtaact tacatgcact ggtaccagcagaagccaggt 120 aaggctccaa agctgctgat ctacagcaca tccaacctgg cttctggtgtgccaagcaga 180 ttctccggaa gcggtagcgg caccgactac accttcacca tcagcagcctccagccagag 240 gatatcgcca cctactactg ccagcagagg agtacttacc cgctcacgttcggccaaggg 300 accaagctcg agatcaaacg g 321 <210> SEQ ID NO 65 <211>LENGTH: 107 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: humanised light chain variable regionvariant <400> SEQUENCE: 65 Gln Ile Val Leu Thr Gln Ser Pro Ser Ser LeuSer Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Ser Ala SerSer Ser Val Thr Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Lys AlaPro Lys Leu Leu Ile Tyr 35 40 45 Ser Thr Ser Asn Leu Ala Ser Gly Val ProSer Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Tyr Thr Phe Thr IleSer Ser Leu Gln Pro Glu 65 70 75 80 Asp Ile Ala Thr Tyr Tyr Cys Gln GlnArg Ser Thr Tyr Pro Leu Thr 85 90 95 Phe Gly Gln Gly Thr Lys Leu Glu IleLys Arg 100 105 <210> SEQ ID NO 66 <211> LENGTH: 22 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: PCR primer for variable region variant <400> SEQUENCE: 66cgtattagtc atcgctatta cc 22 <210> SEQ ID NO 67 <211> LENGTH: 39 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: PCR primer for variable region variant <400> SEQUENCE: 67gttggatgtg ctgtagatcc acagctttgg agccttacc 39 <210> SEQ ID NO 68 <211>LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: PCR primer for variable region variant<400> SEQUENCE: 68 tccgtttgat ctcgagcttg g 21 <210> SEQ ID NO 69 <211>LENGTH: 39 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: PCR primer for variable region variant<400> SEQUENCE: 69 ggtaaggctc caaagctgtg gatctacagc acatccaac 39 <210>SEQ ID NO 70 <211> LENGTH: 321 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: humanisedlight chain variable region variant <400> SEQUENCE: 70 gacatccagatgacccagag cccaagcagc ctgagcgcta gcgtgggtga cagagtgacc 60 atcacgtgtagtgccagctc aagtgtaact tacatgcact ggtaccagca gaagccaggt 120 aaggctccaaagctgtggat ctacagcaca tccaacctgg cttctggtgt gccaagcaga 180 ttctccggaagcggtagcgg caccgactac accttcacca tcagcagcct ccagccagag 240 gatatcgccacctactactg ccagcagagg agtacttacc cgctcacgtt cggccaaggg 300 accaagctcgagatcaaacg g 321 <210> SEQ ID NO 71 <211> LENGTH: 107 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: humanised light chain variable region variant <400>SEQUENCE: 71 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser ValGly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val ThrTyr Met 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu TrpIle Tyr 35 40 45 Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe SerGly Ser 50 55 60 Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser Leu GlnPro Glu 65 70 75 80 Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Thr TyrPro Leu Thr 85 90 95 Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg 100 105<210> SEQ ID NO 72 <211> LENGTH: 64 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: humanisedheavy chain variable region variant insert <400> SEQUENCE: 72 ccttgagtggattgcatgga ttgaccctga gaatggtgac actgagtacg cacctaagtt 60 tcgc 64 <210>SEQ ID NO 73 <211> LENGTH: 68 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: humanised heavy chainvariable region variant insert <400> SEQUENCE: 73 ggccgcgaaa cttaggtgcgtactcagtgt caccattctc agggtcaatc catgcaatcc 60 actcaagg 68 <210> SEQ IDNO 74 <211> LENGTH: 360 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: humanised heavy chainvariable region variant <400> SEQUENCE: 74 gaggtgcagc tgcagcagagcggtccaggt ctcgtacggc ctagccagac cctgagcctc 60 acgtgcaccg catctggcttcaacattaag gacaattaca tgcactgggt gagacagcca 120 cctggacgag gccttgagtggattgcatgg attgaccctg agaatggtga cactgagtac 180 gcacctaagt ttcgcggccgcgtgacaatg ctggcagaca ctagtaagaa ccagttcagc 240 ctgagactca gcagcgtgacagccgccgac accgcggtct attattgtca cgtcctgata 300 tacgccgggt atctggcaatggactactgg ggccaaggga ccctcgtcac cgtgagctcg 360 <210> SEQ ID NO 75 <211>LENGTH: 120 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: humanised heavy chain variable regionvariant <400> SEQUENCE: 75 Glu Val Gln Leu Gln Gln Ser Gly Pro Gly LeuVal Arg Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Ala Ser GlyPhe Asn Ile Lys Asp Asn 20 25 30 Tyr Met His Trp Val Arg Gln Pro Pro GlyArg Gly Leu Glu Trp Ile 35 40 45 Ala Trp Ile Asp Pro Glu Asn Gly Asp ThrGlu Tyr Ala Pro Lys Phe 50 55 60 Arg Gly Arg Val Thr Met Leu Ala Asp ThrSer Lys Asn Gln Phe Ser 65 70 75 80 Leu Arg Leu Ser Ser Val Thr Ala AlaAsp Thr Ala Val Tyr Tyr Cys 85 90 95 His Val Leu Ile Tyr Ala Gly Tyr LeuAla Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser115 120 <210> SEQ ID NO 76 <211> LENGTH: 80 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:humanised heavy chain variable region variant insert <400> SEQUENCE: 76ggccgcgtga caatgctggc agactcaagt aagaaccagg ccagcctgag actcagcagc 60gtgacagccg ccgacaccgc 80 <210> SEQ ID NO 77 <211> LENGTH: 74 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: humanised heavy chain variable region variant insert <400>SEQUENCE: 77 ggtgtcggcg gctgtcacgc tgctgagtct caggctggcc tggttcttacttgagtctgc 60 cagcattgtc acgc 74 <210> SEQ ID NO 78 <211> LENGTH: 360<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: humanised heavy chain variable region variant <400>SEQUENCE: 78 gaggtgcagc tgcagcagag cggtccaggt ctcgtacggc ctagccagaccctgagcctc 60 acgtgcaccg catctggctt caacattaag gacaattaca tgcactgggtgagacagcca 120 cctggacgag gccttgagtg gattggatgg attgaccctg agaatggtgacactgagtac 180 gcacctaagt ttcgcggccg cgtgacaatg ctggcagact caagtaagaaccaggccagc 240 ctgagactca gcagcgtgac agccgccgac accgcggtct attattgtcacgtcctgata 300 tacgccgggt atctggcaat ggactactgg ggccaaggga ccctcgtcaccgtgagctcg 360 <210> SEQ ID NO 79 <211> LENGTH: 120 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: humanised heavy chain variable region variant <400>SEQUENCE: 79 Glu Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Arg Pro SerGln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Ala Ser Gly Phe Asn Ile LysAsp Asn 20 25 30 Tyr Met His Trp Val Arg Gln Pro Pro Gly Arg Gly Leu GluTrp Ile 35 40 45 Gly Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala ProLys Phe 50 55 60 Arg Gly Arg Val Thr Met Leu Ala Asp Ser Ser Lys Asn GlnAla Ser 65 70 75 80 Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala ValTyr Tyr Cys 85 90 95 His Val Leu Ile Tyr Ala Gly Tyr Leu Ala Met Asp TyrTrp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210>SEQ ID NO 80 <211> LENGTH: 360 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: humanisedheavy chain variable region variant <400> SEQUENCE: 80 gaggtgcagctgcagcagag cggtccaggt ctcgtacggc ctagccagac cctgagcctc 60 acgtgcaccgcatctggctt caacattaag gacaattaca tgcactgggt gagacagcca 120 cctggacgaggccttgagtg gattgcatgg attgaccctg agaatggtga cactgagtac 180 gcacctaagtttcgcggccg cgtgacaatg ctggcagact caagtaagaa ccaggccagc 240 ctgagactcagcagcgtgac agccgccgac accgcggtct attattgtca cgtcctgata 300 tacgccgggtatctggcaat ggactactgg ggccaaggga ccctcgtcac cgtgagctcg 360 <210> SEQ IDNO 81 <211> LENGTH: 120 <212> TYPE: PRT <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: humanised heavy chainvariable region variant <400> SEQUENCE: 81 Glu Val Gln Leu Gln Gln SerGly Pro Gly Leu Val Arg Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr CysThr Ala Ser Gly Phe Asn Ile Lys Asp Asn 20 25 30 Tyr Met His Trp Val ArgGln Pro Pro Gly Arg Gly Leu Glu Trp Ile 35 40 45 Ala Trp Ile Asp Pro GluAsn Gly Asp Thr Glu Tyr Ala Pro Lys Phe 50 55 60 Arg Gly Arg Val Thr MetLeu Ala Asp Ser Ser Lys Asn Gln Ala Ser 65 70 75 80 Leu Arg Leu Ser SerVal Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 His Val Leu Ile TyrAla Gly Tyr Leu Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu ValThr Val Ser Ser 115 120 <210> SEQ ID NO 82 <211> LENGTH: 80 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: humanised heavy chain variable region variant insert <400>SEQUENCE: 82 ggccgcgcca caatgctggc agacactagt aagaaccagt tcagcctgagactcagcagc 60 gtgacagccg ccgacaccgc 80 <210> SEQ ID NO 83 <211> LENGTH:74 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: humanised heavy chain variable region variantinsert <400> SEQUENCE: 83 ggtgtcggcg gctgtcacgc tgctgagtct caggctgaactggttcttac tagtgtctgc 60 cagcattgtg gcgc 74 <210> SEQ ID NO 84 <211>LENGTH: 360 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: humanised heavy chain variable region<400> SEQUENCE: 84 gaggtgcagc tgcagcagag cggtccaggt ctcgtacggcctagccagac cctgagcctc 60 acgtgcaccg catctggctt caacattaag gacaattacatgcactgggt gagacagcca 120 cctggacgag gccttgagtg gattggatgg attgaccctgagaatggtga cactgagtac 180 gcacctaagt ttcgcggccg cgccacaatg ctggcagacactagtaagaa ccagttcagc 240 ctgagactca gcagcgtgac agccgccgac accgcggtctattattgtca cgtcctgata 300 tacgccgggt atctggcaat ggactactgg ggccaagggaccctcgtcac cgtgagctcg 360 <210> SEQ ID NO 85 <211> LENGTH: 120 <212>TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: humanised heavy chain variable region <400> SEQUENCE: 85Glu Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Arg Pro Ser Gln 1 5 1015 Thr Leu Ser Leu Thr Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Asn 20 2530 Tyr Met His Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp Ile 35 4045 Gly Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala Pro Lys Phe 50 5560 Arg Gly Arg Ala Thr Met Leu Ala Asp Thr Ser Lys Asn Gln Phe Ser 65 7075 80 Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 8590 95 His Val Leu Ile Tyr Ala Gly Tyr Leu Ala Met Asp Tyr Trp Gly Gln100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> SEQ ID NO 86<211> LENGTH: 80 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: humanised heavy chain variableregion insert <400> SEQUENCE: 86 ggccgcgcca caatgctggc agactcaagtaagaaccagg ccagcctgag actcagcagc 60 gtgacagccg ccgacaccgc 80 <210> SEQID NO 87 <211> LENGTH: 74 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: humanised heavy chainvariable region insert <400> SEQUENCE: 87 ggtgtcggcg gctgtcacgctgctgagtct caggctggcc tggttcttac ttgagtctgc 60 cagcattgtg gcgc 74 <210>SEQ ID NO 88 <211> LENGTH: 360 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: humanisedheavy chain variable region <400> SEQUENCE: 88 gaggtgcagc tgcagcagagcggtccaggt ctcgtacggc ctagccagac cctgagcctc 60 acgtgcaccg catctggcttcaacattaag gacaattaca tgcactgggt gagacagcca 120 cctggacgag gccttgagtggattggatgg attgaccctg agaatggtga cactgagtac 180 gcacctaagt ttcgcggccgcgccacaatg ctggcagact caagtaagaa ccaggccagc 240 ctgagactca gcagcgtgacagccgccgac accgcggtct attattgtca cgtcctgata 300 tacgccgggt atctggcaatggactactgg ggccaaggga ccctcgtcac cgtgagctcg 360 <210> SEQ ID NO 89 <211>LENGTH: 120 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: humanised heavy chain variable region<400> SEQUENCE: 89 Glu Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val ArgPro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Ala Ser Gly Phe AsnIle Lys Asp Asn 20 25 30 Tyr Met His Trp Val Arg Gln Pro Pro Gly Arg GlyLeu Glu Trp Ile 35 40 45 Gly Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu TyrAla Pro Lys Phe 50 55 60 Arg Gly Arg Ala Thr Met Leu Ala Asp Ser Ser LysAsn Gln Ala Ser 65 70 75 80 Leu Arg Leu Ser Ser Val Thr Ala Ala Asp ThrAla Val Tyr Tyr Cys 85 90 95 His Val Leu Ile Tyr Ala Gly Tyr Leu Ala MetAsp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120<210> SEQ ID NO 90 <211> LENGTH: 360 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: humanisedheavy chain variable region <400> SEQUENCE: 90 gaggtgcagc tgcagcagagcggtccaggt ctcgtacggc ctagccagac cctgagcctc 60 acgtgcaccg catctggcttcaacattaag gacaattaca tgcactgggt gagacagcca 120 cctggacgag gccttgagtggattgcatgg attgaccctg agaatggtga cactgagtac 180 gcacctaagt ttcgcggccgcgccacaatg ctggcagact caagtaagaa ccaggccagc 240 ctgagactca gcagcgtgacagccgccgac accgcggtct attattgtca cgtcctgata 300 tacgccgggt atctggcaatggactactgg ggccaaggga ccctcgtcac cgtgagctcg 360 <210> SEQ ID NO 91 <211>LENGTH: 120 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: humanised heavy chain variable region<400> SEQUENCE: 91 Glu Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val ArgPro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Ala Ser Gly Phe AsnIle Lys Asp Asn 20 25 30 Tyr Met His Trp Val Arg Gln Pro Pro Gly Arg GlyLeu Glu Trp Ile 35 40 45 Ala Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu TyrAla Pro Lys Phe 50 55 60 Arg Gly Arg Ala Thr Met Leu Ala Asp Ser Ser LysAsn Gln Ala Ser 65 70 75 80 Leu Arg Leu Ser Ser Val Thr Ala Ala Asp ThrAla Val Tyr Tyr Cys 85 90 95 His Val Leu Ile Tyr Ala Gly Tyr Leu Ala MetAsp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120<210> SEQ ID NO 92 <211> LENGTH: 780 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: humanisedIgG1 sequence <400> SEQUENCE: 92 atgaagttgt ggctgaactg gattttccttgtaacacttt taaatggaat tcagtgtgag 60 gtgcagctgc agcagagcgg tccaggtctcgtacggccta gccagaccct gagcctcacg 120 tgcaccgcat ctggcttcaa cattaaggacaattacatgc actgggtgag acagccacct 180 ggacgaggcc ttgagtggat tggatggattgaccctgaga atggtgacac tgagtacgca 240 cctaagtttc gcggccgcgt gacaatgctggcagacacta gtaagaacca gttcagcctg 300 agactcagca gcgtgacagc cgccgacaccgcggtctatt attgtcacgt cctgatatac 360 gccgggtatc tggcaatgga ctactggggccaagggaccc tcgtcaccgt gagctcggcc 420 tccaccaagg gcccatcggt cttccccctggcaccctcct ccaagagcac ctctgggggc 480 acagcggccc tgggctgcct ggtcaaggactacttccccg aaccggtgac ggtgtcgtgg 540 aactcaggcg ccctgaccag cggcgtgcacaccttcccgg ctgtcctaca gtcctcagga 600 ctctactccc tcagcagcgt ggtgactgtgccctccagca gcttgggcac ccagacctac 660 atctgcaacg tgaatcacaa ccccagcaacaccaaggtcg acaagaaagt tgagcccaaa 720 tcttgtgaca agacgcacac gtgcccgccgtgcccggctc cggaactgct gggtggcccg 780 <210> SEQ ID NO 93 <211> LENGTH:260 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: HuVH1-HuIgG1 Fd heavy chain <400> SEQUENCE: 93Met Lys Leu Trp Leu Asn Trp Ile Phe Leu Val Thr Leu Leu Asn Gly 1 5 1015 Ile Gln Cys Glu Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Arg 20 2530 Pro Ser Gln Thr Leu Ser Leu Thr Cys Thr Ala Ser Gly Phe Asn Ile 35 4045 Lys Asp Asn Tyr Met His Trp Val Arg Gln Pro Pro Gly Arg Gly Leu 50 5560 Glu Trp Ile Gly Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala 65 7075 80 Pro Lys Phe Arg Gly Arg Val Thr Met Leu Ala Asp Thr Ser Lys Asn 8590 95 Gln Phe Ser Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val100 105 110 Tyr Tyr Cys His Val Leu Ile Tyr Ala Gly Tyr Leu Ala Met AspTyr 115 120 125 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser ThrLys Gly 130 135 140 Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser ThrSer Gly Gly 145 150 155 160 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp TyrPhe Pro Glu Pro Val 165 170 175 Thr Val Ser Trp Asn Ser Gly Ala Leu ThrSer Gly Val His Thr Phe 180 185 190 Pro Ala Val Leu Gln Ser Ser Gly LeuTyr Ser Leu Ser Ser Val Val 195 200 205 Thr Val Pro Ser Ser Ser Leu GlyThr Gln Thr Tyr Ile Cys Asn Val 210 215 220 Asn His Asn Pro Ser Asn ThrLys Val Asp Lys Lys Val Glu Pro Lys 225 230 235 240 Ser Cys Asp Lys ThrHis Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 245 250 255 Leu Gly Gly Pro260 <210> SEQ ID NO 94 <211> LENGTH: 918 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: humanisedIgG3 heavy chain Fd sequence <400> SEQUENCE: 94 atgaagttgt ggctgaactggattttcctt gtaacacttt taaatggaat tcagtgtgag 60 gtgcagctgc agcagagcggtccaggtctc gtacggccta gccagaccct gagcctcacg 120 tgcaccgcat ctggcttcaacattaaggac aattacatgc actgggtgag acagccacct 180 ggacgaggcc ttgagtggattggatggatt gaccctgaga atggtgacac tgagtacgca 240 cctaagtttc gcggccgcgtgacaatgctg gcagacacta gtaagaacca gttcagcctg 300 agactcagca gcgtgacagccgccgacacc gcggtctatt attgtcacgt cctgatatac 360 gccgggtatc tggcaatggactactggggc caagggaccc tcgtcaccgt gagctcggct 420 agcaccaagg gcccatcggtcttccccctg gcgccctgct ccaggagcac ctctgggggc 480 acagcggccc tgggctgcctggtcaaggac tacttccccg aaccggtgac ggtgtcgtgg 540 aactcaggcg ccctgaccagcggcgtgcac accttcccgg ctgtcctaca gtcctcagga 600 ctctactccc tcagcagcgtggtgaccgtg ccctccagca gcttgggcac ccagacctac 660 acctgcaacg tgaatcacaagcccagcaac accaaggtgg acaagagagt ggagctgaaa 720 accccactcg gtgacacaactcacacgtgc cctaggtgtc ctgaacctaa atcttgtgac 780 acacctcccc cgtgcccacggtgcccagag cccaaatctt gcgacacgcc cccaccgtgt 840 cccagatgtc ctgaaccaaagagctgtgac actccaccgc cctgcccgag gtgcccagca 900 cctgaactcc tgggaggg 918<210> SEQ ID NO 95 <211> LENGTH: 306 <212> TYPE: PRT <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: humanisedIgG3 heavy chain Fd sequence <400> SEQUENCE: 95 Met Lys Leu Trp Leu AsnTrp Ile Phe Leu Val Thr Leu Leu Asn Gly 1 5 10 15 Ile Gln Cys Glu ValGln Leu Gln Gln Ser Gly Pro Gly Leu Val Arg 20 25 30 Pro Ser Gln Thr LeuSer Leu Thr Cys Thr Ala Ser Gly Phe Asn Ile 35 40 45 Lys Asp Asn Tyr MetHis Trp Val Arg Gln Pro Pro Gly Arg Gly Leu 50 55 60 Glu Trp Ile Gly TrpIle Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala 65 70 75 80 Pro Lys Phe ArgGly Arg Val Thr Met Leu Ala Asp Thr Ser Lys Asn 85 90 95 Gln Phe Ser LeuArg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val 100 105 110 Tyr Tyr CysHis Val Leu Ile Tyr Ala Gly Tyr Leu Ala Met Asp Tyr 115 120 125 Trp GlyGln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 130 135 140 ProSer Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Gly Gly 145 150 155160 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 165170 175 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe180 185 190 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser ValVal 195 200 205 Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Thr CysAsn Val 210 215 220 Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg ValGlu Leu Lys 225 230 235 240 Thr Pro Leu Gly Asp Thr Thr His Thr Cys ProArg Cys Pro Glu Pro 245 250 255 Lys Ser Cys Asp Thr Pro Pro Pro Cys ProArg Cys Pro Glu Pro Lys 260 265 270 Ser Cys Asp Thr Pro Pro Pro Cys ProArg Cys Pro Glu Pro Lys Ser 275 280 285 Cys Asp Thr Pro Pro Pro Cys ProArg Cys Pro Ala Pro Glu Leu Leu 290 295 300 Gly Gly 305 <210> SEQ ID NO96 <211> LENGTH: 705 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: humanised light chain Fdsequence <400> SEQUENCE: 96 atggattttc aagtgcagat tttcagcttc ctgctaatcagtgcttcagt cataatgtcc 60 cgcggccaga tcgtgctgac ccagagccca agcagcctgagcgctagcgt gggtgacaga 120 gtgaccatca cgtgtagtgc cagctcaagt gtaacttacatgcactggta ccagcagaag 180 ccaggtaagg ctccaaagct gctgatctac agcacatccaacctggcttc tggtgtgcca 240 agcagattct ccggaagcgg tagcggcacc gactacaccttcaccatcag cagcctccag 300 ccagaggata tcgccaccta ctactgccag cagaggagtacttacccgct cacgttcggc 360 caagggacca agctcgagat caaacggact gtggctgcaccatctgtctt catcttcccg 420 ccatctgatg agcagttgaa atctggaact gcctctgttgtgtgcctgct gaataacttc 480 tatcccagag aggccaaagt acagtggaag gtggataacgccctccaatc gggtaactcc 540 caggagagtg tcacagagca ggacagcaag gacagcacctacagcctcag cagcaccctg 600 acgctgagca aagcagacta cgagaaacac aaagtctacgcctgcgaagt cacccatcag 660 ggcctgagtt cgcccgtcac aaagagcttc aacaggggagagtgt 705 <210> SEQ ID NO 97 <211> LENGTH: 235 <212> TYPE: PRT <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:humanised light chain Fd sequence <400> SEQUENCE: 97 Met Asp Phe Gln ValGln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser 1 5 10 15 Val Ile Met SerArg Gly Gln Ile Val Leu Thr Gln Ser Pro Ser Ser 20 25 30 Leu Ser Ala SerVal Gly Asp Arg Val Thr Ile Thr Cys Ser Ala Ser 35 40 45 Ser Ser Val ThrTyr Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala 50 55 60 Pro Lys Leu LeuIle Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro 65 70 75 80 Ser Arg PheSer Gly Ser Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile 85 90 95 Ser Ser LeuGln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Arg 100 105 110 Ser ThrTyr Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 115 120 125 ArgThr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 130 135 140Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 145 150155 160 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln165 170 175 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys AspSer 180 185 190 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala AspTyr Glu 195 200 205 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln GlyLeu Ser Ser 210 215 220 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225230 235 <210> SEQ ID NO 98 <211> LENGTH: 705 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:humanised light chain Fd sequence <400> SEQUENCE: 98 atggattttcaagtgcagat tttcagcttc ctgctaatca gtgcttcagt cataatgtcc 60 cgcggcgacatccagatgac ccagagccca agcagcctga gcgctagcgt gggtgacaga 120 gtgaccatcacgtgtagtgc cagctcaagt gtaacttaca tgcactggta ccagcagaag 180 ccaggtaaggctccaaagct gtggatctac agcacatcca acctggcttc tggtgtgcca 240 agcagattctccggaagcgg tagcggcacc gactacacct tcaccatcag cagcctccag 300 ccagaggatatcgccaccta ctactgccag cagaggagta cttacccgct cacgttcggc 360 caagggaccaagctcgagat caaacggact gtggctgcac catctgtctt catcttcccg 420 ccatctgatgagcagttgaa atctggaact gcctctgttg tgtgcctgct gaataacttc 480 tatcccagagaggccaaagt acagtggaag gtggataacg ccctccaatc gggtaactcc 540 caggagagtgtcacagagca ggacagcaag gacagcacct acagcctcag cagcaccctg 600 acgctgagcaaagcagacta cgagaaacac aaagtctacg cctgcgaagt cacccatcag 660 ggcctgagttcgcccgtcac aaagagcttc aacaggggag agtgt 705 <210> SEQ ID NO 99 <211>LENGTH: 235 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: humanised light chain Fd sequence<400> SEQUENCE: 99 Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu IleSer Ala Ser 1 5 10 15 Val Ile Met Ser Arg Gly Asp Ile Gln Met Thr GlnSer Pro Ser Ser 20 25 30 Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile ThrCys Ser Ala Ser 35 40 45 Ser Ser Val Thr Tyr Met His Trp Tyr Gln Gln LysPro Gly Lys Ala 50 55 60 Pro Lys Leu Trp Ile Tyr Ser Thr Ser Asn Leu AlaSer Gly Val Pro 65 70 75 80 Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr AspTyr Thr Phe Thr Ile 85 90 95 Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr TyrTyr Cys Gln Gln Arg 100 105 110 Ser Thr Tyr Pro Leu Thr Phe Gly Gln GlyThr Lys Leu Glu Ile Lys 115 120 125 Arg Thr Val Ala Ala Pro Ser Val PheIle Phe Pro Pro Ser Asp Glu 130 135 140 Gln Leu Lys Ser Gly Thr Ala SerVal Val Cys Leu Leu Asn Asn Phe 145 150 155 160 Tyr Pro Arg Glu Ala LysVal Gln Trp Lys Val Asp Asn Ala Leu Gln 165 170 175 Ser Gly Asn Ser GlnGlu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 180 185 190 Thr Tyr Ser LeuSer Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 195 200 205 Lys His LysVal Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 210 215 220 Pro ValThr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 <210> SEQ ID NO 100<211> LENGTH: 54 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: PCR primer for humanised Fd<400> SEQUENCE: 100 cccagcacct gaactcctgg gaggagcaac aggacacagttatgagaagt acaa 54 <210> SEQ ID NO 101 <211> LENGTH: 50 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: PCR primer for humanised Fd <400> SEQUENCE: 101 gggggtctagattattagta caggtgttcc aggacgtagc tggcaacata 50 <210> SEQ ID NO 102 <211>LENGTH: 46 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: PCR primer for humanised Fd <400>SEQUENCE: 102 gggggagctc ggctagcacc aagggcccat cggtcttccc cctggc 46<210> SEQ ID NO 103 <211> LENGTH: 55 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR primerfor humanised Fd <400> SEQUENCE: 103 ttgtacttct cataactgtg tcctgttgctcctcccagga gttcaggtgc tgggc 55 <210> SEQ ID NO 104 <211> LENGTH: 21<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: PCR primer for humanised Fd <400> SEQUENCE: 104gcctgtgctc aatattgatg g 21 <210> SEQ ID NO 105 <211> LENGTH: 21 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: PCR primer for humanised Fd <400> SEQUENCE: 105 ggagaaagccatatctgcct g 21 <210> SEQ ID NO 106 <211> LENGTH: 30 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: PCR primer <400> SEQUENCE: 106 tcgctattac catggtgatgcggttttggc 30 <210> SEQ ID NO 107 <211> LENGTH: 23 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:PCR primer <400> SEQUENCE: 107 ggctggattc tcagtggcga ctt 23 <210> SEQ IDNO 108 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: PCR primer forhumanised Fd <400> SEQUENCE: 108 cacaacagag gcagttcc 18 <210> SEQ ID NO109 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: PCR primer for humanised Fd<400> SEQUENCE: 109 caccttcacc atcagcagcc 20 <210> SEQ ID NO 110 <211>LENGTH: 19 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: PCR primer for preproHCPB <400>SEQUENCE: 110 ggacctgctg cagagtctg 19 <210> SEQ ID NO 111 <211> LENGTH:47 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: PCR primer for preproHCPB <400> SEQUENCE: 111ggctgcagga attcttatta tagacgaacc cggctatcaa actgagc 47 <210> SEQ ID NO112 <211> LENGTH: 1870 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: expected PCR insert<400> SEQUENCE: 112 aagcttgccg ccaccatgaa gttgtggctg aactggattttccttgtaac acttttaaat 60 ggaattcagt gtgaggtgca gctgcagcag agcggtccaggtctcgtacg gcctagccag 120 accctgagcc tcacgtgcac cgcatctggc ttcaacattaaggacaatta catgcactgg 180 gtgagacagc cacctggacg aggccttgag tggattggatggattgaccc tgagaatggt 240 gacactgagt acgcacctaa gtttcgcggc cgcgtgacaatgctggcaga cactagtaag 300 aaccagttca gcctgagact cagcagcgtg acagccgccgacaccgcggt ctattattgt 360 cacgtcctga tatacgccgg gtatctggca atggactactggggccaagg gaccctcgtc 420 accgtgagct cggctagcac caagggccca tcggtcttccccctggcgcc ctgctccagg 480 agcacctctg ggggcacagc ggccctgggc tgcctggtcaaggactactt ccccgaaccg 540 gtgacggtgt cgtggaactc aggcgccctg accagcggcgtgcacacctt cccggctgtc 600 ctacagtcct caggactcta ctccctcagc agcgtggtgaccgtgccctc cagcagcttg 660 ggcacccaga cctacacctg caacgtgaat cacaagcccagcaacaccaa ggtggacaag 720 agagtggagc tgaaaacccc actcggtgac acaactcacacgtgccctag gtgtcctgaa 780 cctaaatctt gtgacacacc tcccccgtgc ccacggtgcccagagcccaa atcttgcgac 840 acgcccccac cgtgtcccag atgtcctgaa ccaaagagctgtgacactcc accgccctgc 900 ccgaggtgcc cagcacctga actcctggga ggagcaacaggacacagtta tgagaagtac 960 aacaagtggg aaacgataga ggcttggact caacaagtcgccactgagaa tccagccctc 1020 atctctcgca gtgttatcgg aaccacattt gagggacgcgctatttacct cctgaaggtt 1080 ggcaaagctg gacaaaataa gcctgccatt ttcatggactgtggtttcca tgccagagag 1140 tggatttctc ctgcattctg ccagtggttt gtaagagaggctgttcgtac ctatggacgt 1200 gagatccaag tgacagagct tctcgacaag ttagacttttatgtcctgcc tgtgctcaat 1260 attgatggct acatctacac ctggaccaag agccgattttggagaaagac tcgctccacc 1320 catactggat ctagctgcat tggcacagac cccaacagaaattttgatgc tggttggtgt 1380 gaaattggag cctctcgaaa cccctgtgat gaaacttactgtggacctgc cgcagagtct 1440 gaaaaggaga ccaaggccct ggctgatttc atccgcaacaaactctcttc catcaaggca 1500 tatctgacaa tccactcgta ctcccaaatg atgatctacccttactcata tgcttacaaa 1560 ctcggtgaga acaatgctga gttgaatgcc ctggctaaagctactgtgaa agaacttgcc 1620 tcactgcacg gcaccaagta cacatatggc ccgggagctacaacaatcta tccttctgct 1680 gggacttcta aagactgggc ttatgaccaa ggaatcagatattccttcac ctttgaactt 1740 cgagatacag gcagatatgg ctttctcctt ccagaatcccagatccgggc tacctgcgag 1800 gagaccttcc tggcaatcaa gtatgttgcc agctacgtcctggaacacct gtactaataa 1860 tctagagaga 1870 <210> SEQ ID NO 113 <211>LENGTH: 613 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: humanised Fd mutant HCPB sequence<400> SEQUENCE: 113 Met Lys Leu Trp Leu Asn Trp Ile Phe Leu Val Thr LeuLeu Asn Gly 1 5 10 15 Ile Gln Cys Glu Val Gln Leu Gln Gln Ser Gly ProGly Leu Val Arg 20 25 30 Pro Ser Gln Thr Leu Ser Leu Thr Cys Thr Ala SerGly Phe Asn Ile 35 40 45 Lys Asp Asn Tyr Met His Trp Val Arg Gln Pro ProGly Arg Gly Leu 50 55 60 Glu Trp Ile Gly Trp Ile Asp Pro Glu Asn Gly AspThr Glu Tyr Ala 65 70 75 80 Pro Lys Phe Arg Gly Arg Val Thr Met Leu AlaAsp Thr Ser Lys Asn 85 90 95 Gln Phe Ser Leu Arg Leu Ser Ser Val Thr AlaAla Asp Thr Ala Val 100 105 110 Tyr Tyr Cys His Val Leu Ile Tyr Ala GlyTyr Leu Ala Met Asp Tyr 115 120 125 Trp Gly Gln Gly Thr Leu Val Thr ValSer Ser Ala Ser Thr Lys Gly 130 135 140 Pro Ser Val Phe Pro Leu Ala ProCys Ser Arg Ser Thr Ser Gly Gly 145 150 155 160 Thr Ala Ala Leu Gly CysLeu Val Lys Asp Tyr Phe Pro Glu Pro Val 165 170 175 Thr Val Ser Trp AsnSer Gly Ala Leu Thr Ser Gly Val His Thr Phe 180 185 190 Pro Ala Val LeuGln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 195 200 205 Thr Val ProSer Ser Ser Leu Gly Thr Gln Thr Tyr Thr Cys Asn Val 210 215 220 Asn HisLys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Leu Lys 225 230 235 240Thr Pro Leu Gly Asp Thr Thr His Thr Cys Pro Arg Cys Pro Glu Pro 245 250255 Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro Glu Pro Lys 260265 270 Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro Glu Pro Lys Ser275 280 285 Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro Ala Pro Glu LeuLeu 290 295 300 Gly Gly Ala Thr Gly His Ser Tyr Glu Lys Tyr Asn Lys TrpGlu Thr 305 310 315 320 Ile Glu Ala Trp Thr Gln Gln Val Ala Thr Glu AsnPro Ala Leu Ile 325 330 335 Ser Arg Ser Val Ile Gly Thr Thr Phe Glu GlyArg Ala Ile Tyr Leu 340 345 350 Leu Lys Val Gly Lys Ala Gly Gln Asn LysPro Ala Ile Phe Met Asp 355 360 365 Cys Gly Phe His Ala Arg Glu Trp IleSer Pro Ala Phe Cys Gln Trp 370 375 380 Phe Val Arg Glu Ala Val Arg ThrTyr Gly Arg Glu Ile Gln Val Thr 385 390 395 400 Glu Leu Leu Asp Lys LeuAsp Phe Tyr Val Leu Pro Val Leu Asn Ile 405 410 415 Asp Gly Tyr Ile TyrThr Trp Thr Lys Ser Arg Phe Trp Arg Lys Thr 420 425 430 Arg Ser Thr HisThr Gly Ser Ser Cys Ile Gly Thr Asp Pro Asn Arg 435 440 445 Asn Phe AspAla Gly Trp Cys Glu Ile Gly Ala Ser Arg Asn Pro Cys 450 455 460 Asp GluThr Tyr Cys Gly Pro Ala Ala Glu Ser Glu Lys Glu Thr Lys 465 470 475 480Ala Leu Ala Asp Phe Ile Arg Asn Lys Leu Ser Ser Ile Lys Ala Tyr 485 490495 Leu Thr Ile His Ser Tyr Ser Gln Met Met Ile Tyr Pro Tyr Ser Tyr 500505 510 Ala Tyr Lys Leu Gly Glu Asn Asn Ala Glu Leu Asn Ala Leu Ala Lys515 520 525 Ala Thr Val Lys Glu Leu Ala Ser Leu His Gly Thr Lys Tyr ThrTyr 530 535 540 Gly Pro Gly Ala Thr Thr Ile Tyr Pro Ser Ala Gly Thr SerLys Asp 545 550 555 560 Trp Ala Tyr Asp Gln Gly Ile Arg Tyr Ser Phe ThrPhe Glu Leu Arg 565 570 575 Asp Thr Gly Arg Tyr Gly Phe Leu Leu Pro GluSer Gln Ile Arg Ala 580 585 590 Thr Cys Glu Glu Thr Phe Leu Ala Ile LysTyr Val Ala Ser Tyr Val 595 600 605 Leu Glu His Leu Tyr 610 <210> SEQ IDNO 114 <211> LENGTH: 96 <212> TYPE: PRT <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: preproHCPB withC-terminal Leu <400> SEQUENCE: 114 His His Gly Gly Glu His Phe Glu GlyGlu Lys Val Phe Arg Val Asn 1 5 10 15 Val Glu Asp Glu Asn His Ile AsnIle Ile Arg Glu Leu Ala Ser Thr 20 25 30 Thr Gln Ile Asp Phe Trp Lys ProAsp Ser Val Thr Gln Ile Lys Pro 35 40 45 His Ser Thr Val Asp Phe Arg ValLys Ala Glu Asp Thr Val Thr Val 50 55 60 Glu Asn Val Leu Lys Gln Asn GluLeu Gln Tyr Lys Val Leu Ile Ser 65 70 75 80 Asn Leu Arg Asn Val Val GluAla Gln Phe Asp Ser Arg Val Arg Leu 85 90 95 <210> SEQ ID NO 115 <211>LENGTH: 520 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: chimaeric HuIgG3CH1′ Fd construct<400> SEQUENCE: 115 gagctcggct agcaccaagg gcccatcggt cttccccctggcgccctgct ccaggagcac 60 ctctgggggc acagcggccc tgggctgcct ggtcaaggactacttccccg aaccggtgac 120 ggtgtcgtgg aactcaggcg ccctgaccag cggcgtgcacaccttcccgg ctgtcctaca 180 gtcctcagga ctctactccc tcagcagcgt ggtgaccgtgccctccagca gcttgggcac 240 ccagacctac acctgcaacg tgaatcacaa gcccagcaacaccaaggtgg acaagagagt 300 ggagctgaaa accccactcg gtgacacaac tcacacgtgccctaggtgtc ctgaacctaa 360 atcttgtgac acacctcccc cgtgcccacg gtgcccagagcccaaatctt gcgacacgcc 420 cccaccgtgt cccagatgtc ctgaaccaaa gagctgtgacactccaccgc cctgcccgag 480 gtgcccagca cctgaactcc tgggagggta atagcccggg520 <210> SEQ ID NO 116 <211> LENGTH: 31 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR primerfor mutant HCPB <400> SEQUENCE: 116 gttattactc gctgcccaac cagccatggc g31 <210> SEQ ID NO 117 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR primerfor mutant HCPB <400> SEQUENCE: 117 gcagcaggat agattgttgt agc 23 <210>SEQ ID NO 118 <211> LENGTH: 88 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR primerfor mutant HCPB <400> SEQUENCE: 118 ccggaattct tattagttca ggtcctcctcagagatcagc ttctgctcct cgaactcatg 60 gtggtgatgg tggtggtaca ggtgttcc 88<210> SEQ ID NO 119 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR primerfor mutant HCPB <400> SEQUENCE: 119 caatctatcc tgctgctggg acttctaaag 30<210> SEQ ID NO 120 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR primerfor mutant HCPB <400> SEQUENCE: 120 gattgttgta gctcccgggc 20 <210> SEQID NO 121 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: PCR primer for mutantHCPB <400> SEQUENCE: 121 ggagctacaa caatctatcc ttctgctggg 30 <210> SEQID NO 122 <211> LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: PCR primer CME 00971<400> SEQUENCE: 122 acggcaccaa gtacacatat gg 22 <210> SEQ ID NO 123<211> LENGTH: 90 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: PCR primer CME 00971 <400>SEQUENCE: 123 acgagaattc gaccgctctg ctgcagctgc acctcggaac cgccaccgctgccaccgcca 60 gaaccgccac cgtacaggtg ttccaggacg 90 <210> SEQ ID NO 124<211> LENGTH: 2154 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: humanised pre-pro HCPB-linker-Fdsequence <400> SEQUENCE: 124 atgttggcac tcttggttct ggtgactgtg gccctggcatctgctcatca tggtggtgag 60 cactttgaag gcgagaaggt gttccgtgtt aacgttgaagatgaaaatca cattaacata 120 atccgcgagt tggccagcac gacccagatt gacttctggaagccagattc tgtcacacaa 180 atcaaacctc acagtacagt tgacttccgt gttaaagcagaagatactgt cactgtggag 240 aatgttctaa agcagaatga actacaatac aaggtactgataagcaacct gagaaatgtg 300 gtggaggctc agtttgatag ccgggttcgt gcaacaggacacagttatga gaagtacaac 360 aagtgggaaa cgatagaggc ttggactcaa caagtcgccactgagaatcc agccctcatc 420 tctcgcagtg ttatcggaac cacatttgag ggacgcgctatttacctcct gaaggttggc 480 aaagctggac aaaataagcc tgccattttc atggactgtggtttccatgc cagagagtgg 540 atttctcctg cattctgcca gtggtttgta agagaggctgttcgtaccta tggacgtgag 600 atccaagtga cagagcttct cgacaagtta gacttttatgtcctgcctgt gctcaatatt 660 gatggctaca tctacacctg gaccaagagc cgattttggagaaagactcg ctccacccat 720 actggatcta gctgcattgg cacagacccc aacagaaattttgatgctgg ttggtgtgaa 780 attggagcct ctcgaaaccc ctgtgatgaa acttactgtggacctgccgc agagtctgaa 840 aaggagacca aggccctggc tgatttcatc cgcaacaaactctcttccat caaggcatat 900 ctgacaatcc actcgtactc ccaaatgatg atctacccttactcatatgc ttacaaactc 960 ggtgagaaca atgctgagtt gaatgccctg gctaaagctactgtgaaaga acttgcctca 1020 ctgcacggca ccaagtacac atatggcccg ggagctacaacaatctatcc ttctgctggg 1080 acttctaaag actgggctta tgaccaagga atcagatattccttcacctt tgaacttcga 1140 gatacaggca gatatggctt tctccttcca gaatcccagatccgggctac ctgcgaggag 1200 accttcctgg caatcaagta tgttgccagc tacgtcctggaacacctgta cggtggcggt 1260 tctggcggtg gcagcggtgg cggttccgag gtgcagctgcagcagagcgg tccaggtctc 1320 gtacggccta gccagaccct gagcctcacg tgcaccgcatctggcttcaa cattaaggac 1380 aattacatgc actgggtgag acagccacct ggacgaggccttgagtggat tggatggatt 1440 gaccctgaga atggtgacac tgagtacgca cctaagtttcgcggccgcgt gacaatgctg 1500 gcagacacta gtaagaacca gttcagcctg agactcagcagcgtgacagc cgccgacacc 1560 gcggtctatt attgtcacgt cctgatatac gccgggtatctggcaatgga ctactggggc 1620 caagggaccc tcgtcaccgt gagctcggct agcaccaagggcccatcggt cttccccctg 1680 gcgccctgct ccaggagcac ctctgggggc acagcggccctgggctgcct ggtcaaggac 1740 tacttccccg aaccggtgac ggtgtcgtgg aactcaggcgccctgaccag cggcgtgcac 1800 accttcccgg ctgtcctaca gtcctcagga ctctactccctcagcagcgt ggtgaccgtg 1860 ccctccagca gcttgggcac ccagacctac acctgcaacgtgaatcacaa gcccagcaac 1920 accaaggtgg acaagagagt ggagctgaaa accccactcggtgacacaac tcacacgtgc 1980 cctaggtgtc ctgaacctaa atcttgtgac acacctcccccgtgcccacg gtgcccagag 2040 cccaaatctt gcgacacgcc cccaccgtgt cccagatgtcctgaaccaaa gagctgtgac 2100 actccaccgc cctgcccgag gtgcccagca cctgaactcctgggagggta atag 2154 <210> SEQ ID NO 125 <211> LENGTH: 716 <212> TYPE:PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: humanised pre-pro HCPB-linker-Fd sequence <400> SEQUENCE:125 Met Leu Ala Leu Leu Val Leu Val Thr Val Ala Leu Ala Ser Ala His 1 510 15 His Gly Gly Glu His Phe Glu Gly Glu Lys Val Phe Arg Val Asn Val 2025 30 Glu Asp Glu Asn His Ile Asn Ile Ile Arg Glu Leu Ala Ser Thr Thr 3540 45 Gln Ile Asp Phe Trp Lys Pro Asp Ser Val Thr Gln Ile Lys Pro His 5055 60 Ser Thr Val Asp Phe Arg Val Lys Ala Glu Asp Thr Val Thr Val Glu 6570 75 80 Asn Val Leu Lys Gln Asn Glu Leu Gln Tyr Lys Val Leu Ile Ser Asn85 90 95 Leu Arg Asn Val Val Glu Ala Gln Phe Asp Ser Arg Val Arg Ala Thr100 105 110 Gly His Ser Tyr Glu Lys Tyr Asn Lys Trp Glu Thr Ile Glu AlaTrp 115 120 125 Thr Gln Gln Val Ala Thr Glu Asn Pro Ala Leu Ile Ser ArgSer Val 130 135 140 Ile Gly Thr Thr Phe Glu Gly Arg Ala Ile Tyr Leu LeuLys Val Gly 145 150 155 160 Lys Ala Gly Gln Asn Lys Pro Ala Ile Phe MetAsp Cys Gly Phe His 165 170 175 Ala Arg Glu Trp Ile Ser Pro Ala Phe CysGln Trp Phe Val Arg Glu 180 185 190 Ala Val Arg Thr Tyr Gly Arg Glu IleGln Val Thr Glu Leu Leu Asp 195 200 205 Lys Leu Asp Phe Tyr Val Leu ProVal Leu Asn Ile Asp Gly Tyr Ile 210 215 220 Tyr Thr Trp Thr Lys Ser ArgPhe Trp Arg Lys Thr Arg Ser Thr His 225 230 235 240 Thr Gly Ser Ser CysIle Gly Thr Asp Pro Asn Arg Asn Phe Asp Ala 245 250 255 Gly Trp Cys GluIle Gly Ala Ser Arg Asn Pro Cys Asp Glu Thr Tyr 260 265 270 Cys Gly ProAla Ala Glu Ser Glu Lys Glu Thr Lys Ala Leu Ala Asp 275 280 285 Phe IleArg Asn Lys Leu Ser Ser Ile Lys Ala Tyr Leu Thr Ile His 290 295 300 SerTyr Ser Gln Met Met Ile Tyr Pro Tyr Ser Tyr Ala Tyr Lys Leu 305 310 315320 Gly Glu Asn Asn Ala Glu Leu Asn Ala Leu Ala Lys Ala Thr Val Lys 325330 335 Glu Leu Ala Ser Leu His Gly Thr Lys Tyr Thr Tyr Gly Pro Gly Ala340 345 350 Thr Thr Ile Tyr Pro Ser Ala Gly Thr Ser Lys Asp Trp Ala TyrAsp 355 360 365 Gln Gly Ile Arg Tyr Ser Phe Thr Phe Glu Leu Arg Asp ThrGly Arg 370 375 380 Tyr Gly Phe Leu Leu Pro Glu Ser Gln Ile Arg Ala ThrCys Glu Glu 385 390 395 400 Thr Phe Leu Ala Ile Lys Tyr Val Ala Ser TyrVal Leu Glu His Leu 405 410 415 Tyr Gly Gly Gly Ser Gly Gly Gly Ser GlyGly Gly Ser Glu Val Gln 420 425 430 Leu Gln Gln Ser Gly Pro Gly Leu ValArg Pro Ser Gln Thr Leu Ser 435 440 445 Leu Thr Cys Thr Ala Ser Gly PheAsn Ile Lys Asp Asn Tyr Met His 450 455 460 Trp Val Arg Gln Pro Pro GlyArg Gly Leu Glu Trp Ile Gly Trp Ile 465 470 475 480 Asp Pro Glu Asn GlyAsp Thr Glu Tyr Ala Pro Lys Phe Arg Gly Arg 485 490 495 Val Thr Met LeuAla Asp Thr Ser Lys Asn Gln Phe Ser Leu Arg Leu 500 505 510 Ser Ser ValThr Ala Ala Asp Thr Ala Val Tyr Tyr Cys His Val Leu 515 520 525 Ile TyrAla Gly Tyr Leu Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu 530 535 540 ValThr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 545 550 555560 Ala Pro Cys Ser Arg Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 565570 575 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser580 585 590 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu GlnSer 595 600 605 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro SerSer Ser 610 615 620 Leu Gly Thr Gln Thr Tyr Thr Cys Asn Val Asn His LysPro Ser Asn 625 630 635 640 Thr Lys Val Asp Lys Arg Val Glu Leu Lys ThrPro Leu Gly Asp Thr 645 650 655 Thr His Thr Cys Pro Arg Cys Pro Glu ProLys Ser Cys Asp Thr Pro 660 665 670 Pro Pro Cys Pro Arg Cys Pro Glu ProLys Ser Cys Asp Thr Pro Pro 675 680 685 Pro Cys Pro Arg Cys Pro Glu ProLys Ser Cys Asp Thr Pro Pro Pro 690 695 700 Cys Pro Arg Cys Pro Ala ProGlu Leu Leu Gly Gly 705 710 715 <210> SEQ ID NO 126 <211> LENGTH: 42<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: PCR primer for mutant HCPB <400> SEQUENCE: 126tatataaagc ttgccgccac catgggccac acacggaggc ag 42 <210> SEQ ID NO 127<211> LENGTH: 45 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: PCR primer for mutant HCPB <400>SEQUENCE: 127 actccaccag cttcacctcg ttatcaggaa aatgctcttg cttgg 45 <210>SEQ ID NO 128 <211> LENGTH: 45 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR primerfor mutant HCPB <400> SEQUENCE: 128 agagcatttt cctgataacg aggtgaagctggtggagtct ggagg 45 <210> SEQ ID NO 129 <211> LENGTH: 40 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: PCR primer for mutant HCPB <400> SEQUENCE: 129 ccaggcatcccagggtcacc atggagttag tttgggcagc 40 <210> SEQ ID NO 130 <211> LENGTH:1446 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: full-length human B7.1-murine ASB7 Fd fusion<221> NAME/KEY: CDS <222> LOCATION: (16)..(1434) <223> OTHERINFORMATION: <400> SEQUENCE: 130 aagcttgccg ccacc atg ggc cac aca cggagg cag gga aca tca cca tcc 51 Met Gly His Thr Arg Arg Gln Gly Thr SerPro Ser 1 5 10 aag tgt cca tac ctc aat ttc ttt cag ctc ttg gtg ctg gctggt ctt 99 Lys Cys Pro Tyr Leu Asn Phe Phe Gln Leu Leu Val Leu Ala GlyLeu 15 20 25 tct cac ttc tgt tca ggt gtt atc cac gtg acc aag gaa gtg aaagaa 147 Ser His Phe Cys Ser Gly Val Ile His Val Thr Lys Glu Val Lys Glu30 35 40 gtg gca acg ctg tcc tgt ggt cac aat gtt tct gtt gaa gag ctg gca195 Val Ala Thr Leu Ser Cys Gly His Asn Val Ser Val Glu Glu Leu Ala 4550 55 60 caa act cgc atc tac tgg caa aag gag aag aaa atg gtg ctg act atg243 Gln Thr Arg Ile Tyr Trp Gln Lys Glu Lys Lys Met Val Leu Thr Met 6570 75 atg tct ggg gac atg aat ata tgg ccc gag tac aag aac cgg acc atc291 Met Ser Gly Asp Met Asn Ile Trp Pro Glu Tyr Lys Asn Arg Thr Ile 8085 90 ttt gat atc act aat aac ctc tcc att gtg atc ctg gct ctg cgc cca339 Phe Asp Ile Thr Asn Asn Leu Ser Ile Val Ile Leu Ala Leu Arg Pro 95100 105 tct gac gag ggc aca tac gag tgt gtt gtt ctg aag tat gaa aaa gac387 Ser Asp Glu Gly Thr Tyr Glu Cys Val Val Leu Lys Tyr Glu Lys Asp 110115 120 gct ttc aag cgg gaa cac ctg gct gaa gtg acg tta tca gtc aaa gct435 Ala Phe Lys Arg Glu His Leu Ala Glu Val Thr Leu Ser Val Lys Ala 125130 135 140 gac ttc cct aca cct agt ata tct gac ttt gaa att cca act tctaat 483 Asp Phe Pro Thr Pro Ser Ile Ser Asp Phe Glu Ile Pro Thr Ser Asn145 150 155 att aga agg ata att tgc tca acc tct gga ggt ttt cca gag cctcac 531 Ile Arg Arg Ile Ile Cys Ser Thr Ser Gly Gly Phe Pro Glu Pro His160 165 170 ctc tcc tgg ttg gaa aat gga gaa gaa tta aat gcc atc aac acaaca 579 Leu Ser Trp Leu Glu Asn Gly Glu Glu Leu Asn Ala Ile Asn Thr Thr175 180 185 gtt tcc caa gat cct gaa act gag ctc tat gct gtt agc agc aaactg 627 Val Ser Gln Asp Pro Glu Thr Glu Leu Tyr Ala Val Ser Ser Lys Leu190 195 200 gat ttc aat atg aca acc aac cac agc ttc atg tgt ctc atc aagtat 675 Asp Phe Asn Met Thr Thr Asn His Ser Phe Met Cys Leu Ile Lys Tyr205 210 215 220 gga cat tta aga gtg aat cag acc ttc aac tgg aat aca accaag caa 723 Gly His Leu Arg Val Asn Gln Thr Phe Asn Trp Asn Thr Thr LysGln 225 230 235 gag cat ttt cct gat aac gag gtg aag ctg gtg gag tct ggagga ggc 771 Glu His Phe Pro Asp Asn Glu Val Lys Leu Val Glu Ser Gly GlyGly 240 245 250 ttg gta cag cct ggg ggt tct ctg aga ctc tcc tgt gca acttct ggg 819 Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Thr SerGly 255 260 265 ttc acc ttc act gat tac tac atg aac tgg gtc cgc cag cctcca gga 867 Phe Thr Phe Thr Asp Tyr Tyr Met Asn Trp Val Arg Gln Pro ProGly 270 275 280 aag gca ctt gag tgg ttg ggt ttt att gga aac aaa gct aatggt tac 915 Lys Ala Leu Glu Trp Leu Gly Phe Ile Gly Asn Lys Ala Asn GlyTyr 285 290 295 300 aca aca gag tac agt gca tct gtg aag ggt cgg ttc accatc tcc aga 963 Thr Thr Glu Tyr Ser Ala Ser Val Lys Gly Arg Phe Thr IleSer Arg 305 310 315 gac aaa tcc caa agc atc ctc tat ctt caa atg aac accctg aga gct 1011 Asp Lys Ser Gln Ser Ile Leu Tyr Leu Gln Met Asn Thr LeuArg Ala 320 325 330 gag gac agt gcc act tat tac tgt aca aga gat agg gggcta cgg ttc 1059 Glu Asp Ser Ala Thr Tyr Tyr Cys Thr Arg Asp Arg Gly LeuArg Phe 335 340 345 tac ttt gac tac tgg ggc caa ggc acc act ctc aca gtctcc tca gcc 1107 Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val SerSer Ala 350 355 360 aaa acg aca ccc cca tct gtc tat cca ctg gcc cct ggatct gct gcc 1155 Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly SerAla Ala 365 370 375 380 caa act aac tcc atg gtg acc ctg gga tgc ctg gtcaag ggc tat ttc 1203 Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val LysGly Tyr Phe 385 390 395 cct gag cca gtg aca gtg acc tgg aac tct gga tctctg tcc agc ggt 1251 Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser LeuSer Ser Gly 400 405 410 gtg cac acc ttc cca gct gtc ctg cag tct gac ctctac act ctg agc 1299 Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu TyrThr Leu Ser 415 420 425 agc tca gtg act gtc ccc tcc agc acc tgg ccc agcgag acc gtc acc 1347 Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser GluThr Val Thr 430 435 440 tgc aac gtt gcc cac ccg gcc agc agc acc aag gtggac aag aaa att 1395 Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val AspLys Lys Ile 445 450 455 460 gtg ccc agg gat tgt ggt tgt aag cct tgc atatgt aca tagtaagaat tc 1446 Val Pro Arg Asp Cys Gly Cys Lys Pro Cys IleCys Thr 465 470 <210> SEQ ID NO 131 <211> LENGTH: 473 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: full-length human B7.1-murine ASB7 Fd fusion <400>SEQUENCE: 131 Met Gly His Thr Arg Arg Gln Gly Thr Ser Pro Ser Lys CysPro Tyr 1 5 10 15 Leu Asn Phe Phe Gln Leu Leu Val Leu Ala Gly Leu SerHis Phe Cys 20 25 30 Ser Gly Val Ile His Val Thr Lys Glu Val Lys Glu ValAla Thr Leu 35 40 45 Ser Cys Gly His Asn Val Ser Val Glu Glu Leu Ala GlnThr Arg Ile 50 55 60 Tyr Trp Gln Lys Glu Lys Lys Met Val Leu Thr Met MetSer Gly Asp 65 70 75 80 Met Asn Ile Trp Pro Glu Tyr Lys Asn Arg Thr IlePhe Asp Ile Thr 85 90 95 Asn Asn Leu Ser Ile Val Ile Leu Ala Leu Arg ProSer Asp Glu Gly 100 105 110 Thr Tyr Glu Cys Val Val Leu Lys Tyr Glu LysAsp Ala Phe Lys Arg 115 120 125 Glu His Leu Ala Glu Val Thr Leu Ser ValLys Ala Asp Phe Pro Thr 130 135 140 Pro Ser Ile Ser Asp Phe Glu Ile ProThr Ser Asn Ile Arg Arg Ile 145 150 155 160 Ile Cys Ser Thr Ser Gly GlyPhe Pro Glu Pro His Leu Ser Trp Leu 165 170 175 Glu Asn Gly Glu Glu LeuAsn Ala Ile Asn Thr Thr Val Ser Gln Asp 180 185 190 Pro Glu Thr Glu LeuTyr Ala Val Ser Ser Lys Leu Asp Phe Asn Met 195 200 205 Thr Thr Asn HisSer Phe Met Cys Leu Ile Lys Tyr Gly His Leu Arg 210 215 220 Val Asn GlnThr Phe Asn Trp Asn Thr Thr Lys Gln Glu His Phe Pro 225 230 235 240 AspAsn Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro 245 250 255Gly Gly Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe Thr 260 265270 Asp Tyr Tyr Met Asn Trp Val Arg Gln Pro Pro Gly Lys Ala Leu Glu 275280 285 Trp Leu Gly Phe Ile Gly Asn Lys Ala Asn Gly Tyr Thr Thr Glu Tyr290 295 300 Ser Ala Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Lys SerGln 305 310 315 320 Ser Ile Leu Tyr Leu Gln Met Asn Thr Leu Arg Ala GluAsp Ser Ala 325 330 335 Thr Tyr Tyr Cys Thr Arg Asp Arg Gly Leu Arg PheTyr Phe Asp Tyr 340 345 350 Trp Gly Gln Gly Thr Thr Leu Thr Val Ser SerAla Lys Thr Thr Pro 355 360 365 Pro Ser Val Tyr Pro Leu Ala Pro Gly SerAla Ala Gln Thr Asn Ser 370 375 380 Met Val Thr Leu Gly Cys Leu Val LysGly Tyr Phe Pro Glu Pro Val 385 390 395 400 Thr Val Thr Trp Asn Ser GlySer Leu Ser Ser Gly Val His Thr Phe 405 410 415 Pro Ala Val Leu Gln SerAsp Leu Tyr Thr Leu Ser Ser Ser Val Thr 420 425 430 Val Pro Ser Ser ThrTrp Pro Ser Glu Thr Val Thr Cys Asn Val Ala 435 440 445 His Pro Ala SerSer Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp 450 455 460 Cys Gly CysLys Pro Cys Ile Cys Thr 465 470

1. An anti-CEA antibody (“806.077 Ab”) comprising complementaritydetermining regions (CDRs) in which the CDRs comprise the followingsequences: a) heavy chain CDRI DNYMH (SEQ ID NO: 29) CDR2 WIDPENGDTEYAPKFRG (SEQ ID NO: 31) CDR3 LIYAGYLAMD Y(SEQ ID NO: 32); and b) lightchain CDRI SASSSVTYMH (SEQ ID NO: 26) CDR2 STSNLAS (SEQ ID NO: 27) CDR3QQRSTYPLT (SEQ ID NO: 28).


2. An antibody according to claim 1 in which the heavy chain CDRs 1 and3 are further defined as: CDR1 FNIKDNYMH (SEQ ID NO: 30); and CDR3HVLIYAGYLA MDY (SEQ ID NO: 33).


3. An antibody according to claim 1 comprising the following, optionallyhumanised, structure: a heavy chain variable region sequence (SEQ ID NO:11) EVQLQQSGAE LVRSGASVKL SCTASGFNIK DNYMHWVKQR 40 PEQGLEWIAW IDPENGDTEYAPKFRGKATL TADSSSNTAY 80 LHLSSLTSED TAVYYCHVLI YAGYLAMDYW GQGTSVAVSS 120and; a light chain variable region sequence (SEQ ID NO: 9): DIELTQSPAIMSASPGEKVT ITCSASSSVT YMHWFQQKPG 40 TSPKLWIYST SNLASGVPAR FSGSGSGTSYSLTISRMEAE 80 DAATYYCQQR STYPLTFGAG TKLELKRA
 108.


4. A humanised antibody according to claim 3 comprising at least one ofthe following sequences: a heavy chain variable region sequence which isVH1 (SEQ ID NO: 55); a light chain variable region sequence which is VK4(SEQ ID NO: 71); a human CH1 heavy chain IgG3 constant region; a humankappa light chain CL region; and a human IgG3 hinge region; optionallyin the form of a F(ab′)₂ fragment.
 5. A conjugate comprising an antibodyaccording to any preceding claim and an effector moiety.
 6. A conjugateaccording to claim 5 in which the effector moiety is selected from anyone of the following: a) an enzyme suitable for use in an ADEPT system;b) CPG2; c) [G251T,D253K]HCPB; d) [A248S,G251T,D253K]HCPB; e) aco-stimulatory molecule; f) extracellular domain of B7; g) extracellulardomain of human B7.1; and h) extracellular domain of human B7.2;optionally in the form of a fusion protein.
 7. A conjugate according toclaim 6 which is a fusion protein selected from any one of the followingconjugates, (sequences being listed in N terminus to C terminusdirection): a) a humanised 806.077 F(ab′)₂-{[A248S,G251T,D253K]HCPB}₂fusion comprising: an antibody Fd′ chain of structure VH1(SEQ ID NO:55)/CH1 constant region from IgG3/hinge region from IgG3; the Fd′ chainbeing fused via its C terminus to the N terminus of[A248S,G251T,D253K]HCPB; and an antibody light chain of formula VK4(SEQID NO: 71)/CL region from kappa light chain; b){[A248S,G251T,D253K]HCPB}₂-humanised 806.077 F(ab′)₂ fusion comprising:[A248S,G251T,D253K]HCPB; the HCPB being fused at its C terminus, via a(GGGS)₃ linker, to the N terminus of an antibody Fd′ chain of structureVH1 (SEQ ID NO: 55)/CH1 constant region from IgG3/hinge region fromIgG3; and an antibody light chain of formula VK4(SEQ ID NO: 71)/CLregion from kappa light chain; and c) a (human B7.1 extracellulardomain)₂ - humanised 806.077 F(ab′)₂ fusion comprising: human B7.1extracellular domain; the B7.1 being fused at its C terminus to the Nterminus of an antibody Fd′ chain of structure VH1(SEQ ID NO: 55)/CH1constant region from IgG3/hinge region from IgG3; and an antibody lightchain of structure VK4(SEQ ID NO: 71)/CL region from kappa light chain.8. A polynucleotide sequence capable of encoding a polypeptide of anantibody or a conjugate as defined in any preceding claim.
 9. A vectorcomprising a polynucleotide as defined in claim
 8. 10. A host celltransformed with a polynucleotide sequence as defined in claim 8 or atransgenic non-human animal or transgenic plant developed from the hostcell.
 11. Hybridoma 806.077 deposited as ECACC deposit no.
 96022936. 12.A pharmaceutical composition comprising a conjugate as defined in anypreceding claim in association with a pharmaceutically-acceptablediluent or carrier, optionally in a form suitable for intravenousadministration.
 13. A conjugate as described in any preceding claim foruse as a medicament.
 14. A method of making an antibody or a conjugateas defined in any preceding claim which comprises: a) subjecting a hostcell, a transgenic non-human mammal or a transgenic plant as defined inclaim 10, or the hybridoma of claim 11, to conditions conducive toexpression, and optionally secretion, of the antibody or conjugate; andoptionally b) at least partially purifying the antibody or conjugate.15. A method of treatment of a human or animal in need of such treatmentwhich comprises administration to a human or animal of apharmaceutically effective amount of a conjugate as defined in anypreceding claim.